LSD1 Research Articles

Upregulation of lysine-specific demethylase 6B aggravatesinflammatory pain through H3K27me3 demethylation-dependent production of TNF-α in the dorsal root ganglia and spinal dorsal horn in rats.

Lysine-specific demethylase 6B (KDM6B) serves as a key mediator of gene transcription. It regulates expression of proinflammatory cytokines and chemokines in variety of diseases. Herein, the role and the underlying mechanisms of KDM6B in inflammatory pain were studied. The inflammatory pain was conducted by intraplantar injection of complete Freund's adjuvant (CFA) in rats. Immunofluorescence, Western blotting, qRT-PCR, and chromatin immunoprecipitation (ChIP)-PCR were performed to investigate the underlying mechanisms. CFA injection led to upregulation of KDM6B and decrease in the level of H3K27me3 in the dorsal root ganglia (DRG) and spinal dorsal horn. The mechanical allodynia and thermal hyperalgesia following CFA were alleviated by the treatment of intrathecal injection of GSK-J4, and by microinjection of AAV-EGFP-KDM6B shRNA in the sciatic nerve or in lumbar 5 dorsal horn. The increased production of tumor necrosis factor-α (TNF-α) following CFA in the DRGs and dorsal horn was inhibited by these treatments. ChIP-PCR showed that CFA-induced increased binding of nuclear factor κB with TNF-α promoter was repressed by the treatment of microinjection of AAV-EGFP-KDM6B shRNA. These results suggest that upregulated KDM6B via facilitating TNF-α expression in the DRG and spinal dorsal horn aggravates inflammatory pain.

Targeting intracellular proteins with cell type-specific functions for cancer immunotherapy

Abstract Immune checkpoint inhibitors (ICIs) use antibodies that block cell surface immune checkpoint proteins with great efficacy in treating immunogenic or “immune hot” tumors such as melanoma, kidney, and lung adenocarcinoma. ICIs have limited response rates to other non-immunogenic cancers. The tumor microenvironment (TME) consists of many cell types that collectively promote tumor progression. Cancer therapeutics are commonly designed to target one molecule in one defined cell type. There is growing evidence that long-term therapeutic responses require the targeting of cancer cells and tumor-promoting populations within the TME. The question remains whether we can identify targetable molecules/pathways that are critical for multiple cell types. Here, we will discuss several molecular targets that may fit a “two or multiple birds, one stone” model, including the B-cell lymphoma-2 (BCL-2) family pro-survival factors, transcriptional factors including signal transducer and activator of transcription 3, the nuclear receptor 4A family (NR4A1, NR4A2, and NR4A3), as well as epigenetic regulators such as bromodomain and extra-terminal (BET) family proteins, histone deacetylase family, SET domain bifurcated histone lysine methyltransferase 1 (SETDB1), and lysine-specific demethylase 1 (LSD1/KDM1A). We will focus on the rationale of these targets in immune modulation, as well as the strategies for targeting these important proteins for cancer therapy.

MiR-29c-3p Accelerates Mucosal Injury in Dextran Sodium Sulfate-induced Ulcerative Colitis Mice through the KDM6B/ H3K27me3/LDHA axis.

Ulcerative colitis (UC) is an inflammatory intestinal disorder featured by mucosal injury. MicroRNAs (miRNAs) play a role in the pathogenesis underlying UC. This study was conducted to investigate the role of miR-29c-3p in a dextran sodium sulfate (DSS)-induced UC mouse model and provide targets for UC treatment. The UC mouse model was established by DSS induction. The expression levels of miR-29c-3p, lysine-specific demethylase 6B (KDM6B), zonula occludens-1 (ZO-1), Occludin, and lactate dehydrogenase A (LDHA) were detected by real-time quantitative polymerase chain reaction or Western blot assays. The mucosal injury was evaluated by disease activity index (DAI), colon length, Hematoxylin-Eosin staining, and fluorescein isothiocyanate-glucan permeability test. The binding between miR-29c-3p and KDM6B and the occupation of KDM6B or trimethylated H3 lysine 27 (H3K27me3) on the LDHA promoter were analyzed by the dual-luciferase and chromatin-immunoprecipitation assays. miR-29c-3p was downregulated while KDM6B and LDHA were upregulated in DSS mice. miR-29c-3p overexpression reduced DAI and inflammatory cell infiltration while increasing colon length, intestinal permeability, and levels of ZO-1 and Occludin. miR-29c-3p inhibited KDM6B expression and increased H3K27me3 occupation on the LDHA promoter, thus inhibiting LDHA transcription. Overexpression of KDM6B or LDHA averted the protective role of miR-29c-3p upregulation in mucosal injury. miR-29c-3p limited KDM6B expression and increased the H3K27me3 occupation on the LDHA promoter to enhance LDHA transcription, moderating mucosal injury and delaying UC progression.

Iadademstat in combination with gilteritinib for patients with mutated FLT3 relapsed/refractory acute myeloid leukemia.

TPS7075 Background: Epigenetic dysregulation is a hallmark of acute myeloid leukemia (AML) and is caused by recurrent translocations and/or mutations in chromatin regulators and transcription factors, resulting in myeloid differentiation blockade and leukemic stem cell renewal. Accordingly, about 70% of recurring mutations in AML patients (pts) target regulators of gene expression, underscoring the potential of epigenetic therapies to change the disease natural history. Iadademstat (iada/ORY-1001) is a specific, oral, potent, covalent inhibitor of the epigenetic Lysine-Specific Demethylase 1 (LSD1/KDMA1) enzyme. In preclinical and clinical studies iada decreased leukemic stem cell survival and induced macrophage/monocytic differentiation of blasts. ALICE, a Ph2 study of iada in combination with azacitidine (aza), showed high complete remission (CR/CRi) rates and durable responses in treatment naïve, unfit AML pts without exacerbating the toxicity profile of aza (Salamero et al., Oral at ASH 2022). Despite improvements in AML therapy, relapsed and refractory (R/R) cases are frequent and contribute to the death of more than 50% of pts, particularly in those subpopulations with higher risk genetics, the largest of which (up to 30-40% AML pts) harbors fms-like tyrosine kinase 3 mutations (FLT3mut+). Use of the FLT3 inhibitor (FLT3i) gilteritinib as monotherapy for R/R pts resulted in improved outcomes but the duration of remission achieved is transient and often brief (CR rate: 20%; EFS: 2.8 months) per the ADMIRAL Ph3 study (Perl, et al., NEJM 2019). Preclinically, iada has marked synergy with FLT3i, particularly gilteritinib, in FLT3 wild-type and FLT3 mut+ AML cells and in derived cell lines resistant to venetoclax, azacitidine and FLT3is. Methods: Adult pts with ECOG 0-2 and FLT3 mut+ R/R AML, after 1 or 2 prior lines of therapy, will be enrolled. In escalation (3+3), up to 18 pts will receive iada at 75 to 150 ug, orally, in 5 days ON-2 days OFF schedule, with gilteritinib SoC. Up to approximate 14 pts will be expanded from pharmacologically active dose/s (per Project OPTIMUS). Primary endpoints of the study are safety and RP2D determination. Bayesian posterior probability efficacy monitoring will be performed periodically for each dose cohort. Bayesian efficacy futility and early stopping boundary will be applied. Posterior probability criterion (Prob (CR > 0.3) ≥ 0.60) at the end of the study will warrant further research. Safety in expansion will be evaluated continuously with a Bayesian stopping rule. Secondary endpoints include OS, EFS, CR, CR/CRh, ORR, TTR, DoR, and transfusion rate. Exploratory endpoints include PK, PD, MRD and gene mutational analysis. The study will run in 15 US sites. Additional sites will be added for a subsequent Phase 2 randomized controlled double-blinded FRIDA 2 study to assess the efficacy of this combination in R/R FLT3 mut+ AML. Clinical trial information: NCT05546580 .

Discovery of new tetrahydroisoquinolines as potent and selective LSD1 inhibitors for the treatment of MLL-rearranged leukemia

Histone lysine-specific demethylase 1 (LSD1) is a promising target for cancer therapy. Here, we performed the design, synthesis, and extensive structure-activity relationship (SAR) studies based on our previously discovered natural LSD1 inhibitor, higenamine. We found that the tetracyclic tetrahydroisoquinoline FY-21 is a potent and selective inhibitor of LSD1 (IC50 = 340 nM). FY-21 inhibited leukemia cell proliferation and colony formation and increased the level of p53 expression. Meanwhile, FY-21 reduced the mRNA levels of the transcription factors HOXA9 and MEIS1. Furthermore, FY-21 significantly induced leukemia cell differentiation. In vivo studies showed that FY-21 prolonged the survival rate of leukemia mice. Collectively, FY-21 is a potent, selective LSD1 inhibitor and can serve as a lead compound for the development of novel and highly effective LSD1 inhibitors for AML treatment.

Targeting the YAP/TAZ mechanotransducers in solid tumour therapeutics.

Targeting the YAP/TAZ mechanotransducers in solid tumour therapeutics.

Pharmacological inhibition of LSD1 suppresses growth of hepatocellular carcinoma by inducing GADD45B.

Lysine-specific histone demethylase 1 (LSD1) is an attractive target for malignancies therapy. Nevertheless, its role in hepatocellular carcinoma (HCC) progression and the potential of its inhibitor in HCC therapy remains unclear. Here, we show that LSD1 overexpression in human HCC tissues is associated with HCC progression and poor patient survival. ZY0511, a highly selective and potent inhibitor of LSD1, suppressed human HCC cell proliferation in vitro and tumor growth in cell-derived and patient-derived HCC xenograft models in vivo. Mechanistically, ZY0511 induced mRNA expression of growth arrest and DNA damage-inducible gene 45beta (GADD45B) by inducing histone H3 at lysine 4 (H3K4) methylation at the promoter of GADD45B, a novel target gene of LSD1. In human HCC tissues, LSD1 level was correlated with a decreased level of GADD45B, which was associated with HCC progression and predicted poor patient survival. Moreover, co-administration of ZY0511 and DTP3, which specifically enhanced the pro-apoptotic effect of GADD45B, effectively inhibited HCC cell proliferation both in vitro and in vivo. Collectively, our study revealed the potential value of LSD1 as a promising target of HCC therapy. ZY0511 is a promising candidate for HCC therapy through upregulating GADD45B, thereby providing a novel combinatorial strategy for treating HCC.

Discovery of novel tranylcypromine-based derivatives as LSD1 inhibitors for gastric cancer treatment.

As an important epigenetic regulator, histone lysine specific demethylase 1 (LSD1) has become an attractive target for the discovery of anticancer agents. In this work, a series of tranylcypromine-based derivatives were designed and synthesized. Among them, compound 12u exhibited the most potent inhibitory potency on LSD1 (IC50=25.3nM), and also displayed good antiproliferative effects on MGC-803, KYSE450 and HCT-116cells with IC50 values of 14.3, 22.8 and 16.3μM, respectively. Further studies revealed that compound 12u could directly act on LSD1 and inhibit LSD1 in MGC-803cells, thereby significantly increasing the expression levels of mono-/bi-methylation of H3K4 and H3K9. In addition, compound 12u could induce apoptosis and differentiation, inhibit migration and cell stemness in MGC-803cells. All these findings suggested that compound 12u was an active tranylcypromine-based derivative as a LSD1 inhibitor that inhibited gastric cancer.

Role of histone methylation in skin cancers: Histone methylation-modifying enzymes as a new class of targets for skin cancer treatment.

Histone methylation, one of the most prominent epigenetic modifications, plays a vital role in gene transcription, and aberrant histone methylation levels cause tumorigenesis. Histone methylation is a reversible enzyme-dependent reaction, and histone methyltransferases and demethylases are involved in this reaction. This review addresses the biological and clinical relevance of these histone methylation-modifying enzymes for skin cancer. In particular, the roles of histone lysine methyltransferases, histone arginine methyltransferase, lysine-specific demethylases, and JmjC demethylases in skin cancer are discussed in detail. In addition, we summarize the efficacy of several epigenetic inhibitors targeting histone methylation-modifying enzymes in cutaneous cancers, such as basal cell carcinoma (BCC), squamous cell carcinoma (SCC), and melanoma. In conclusion, we propose histone methylation-modifying enzymes as novel targets for next-generation pharmaceuticals in the treatment of skin cancers and further provide a rationale for the development of epigenetic drugs (epidrugs) that target specific histone methylases/demethylases in cutaneous tumors.

Formaldehyde dehydrogenase-derived formate contributes to cardioprotection in the female heart

Ischemic heart disease (IHD) is a leading cause of death for men and women in the US, often manifesting as myocardial ischemia/reperfusion (I/R) injury. The risk for IHD, however, is considerably lower in premenopausal women vs. age-matched men and postmenopausal women, suggesting an estrogen (E2)-dependent protective mechanism. Previously, we found female hearts to contain two-fold more formaldehyde (FA) vs. males, but also higher activity of FA dehydrogenase (FDH), which detoxifies FA to formate. Importantly, we found that FDH inhibition or knockout exacerbates I/R injury in female hearts, potentially due to a lack of FDH-derived formate, yet mechanistic data is lacking. The objective of this study is to investigate a potential role for E2 in the regulation of FA production and FDH-mediated metabolism of FA to formate. We also aim to understand the link between FA metabolism and protection in female hearts. We hypothesize that E2 enhances FA metabolism in female hearts to increase formate production and contribute to protection from I/R injury. Our findings show a significant decrease in FDH activity in ovariectomized (OVX) female hearts, but also decreased FA vs. intact females, suggesting that FA production and metabolism are E2-dependent. To determine differences in enzymes for FA production, western blots were performed in lysates from male, female, and OVX mouse hearts. Serine hydroxymethyltranserfases 1 and 2 were more highly expressed in females vs. males. Lysine-specific demethylase 1 expression was significantly higher in female hearts vs. OVX and males. To further assess a role for formate in I/R injury, male and female hearts were Langendorff-perfused in the presence or absence of 30 μM formate. While formate treatment reduced infarct size in both sexes, this effect was more pronounced and significant in males (male: 36.6 +/- 5.45% vs. 10.3 +/- 1.46%, n = 9-10, p < 0.001; female: 19.9 +/- 1.57% vs. 15.8 +/- 1.70%, n = 9-10, p = 0.11). We also found more formate in plasma from females vs. males potentially explaining the blunted protection observed with formate in females. More importantly, we found that formate served as an effective rescue for FDH knockout female hearts, significantly reducing infarct size following I/R injury (60.0 +/- 3.55% vs. 40.2 +/- 2.79%, n = 6, p < 0.01). Taken together, our findings demonstrate that formate protects against I/R injury and further suggest that FDH-derived formate is a critical component of female-specific cardioprotection. As FDH is essential for female-specific cardioprotection from I/R injury, we will next determine whether formate rescues OVX hearts to determine its therapeutic potential for postmenopausal women. NHLBI 5T32ES007141 (HG), AHA 903435 (HG), NHLBI 1F31HL165920-01 (HG), RO1HL13649 (MK) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Nutrient regulation of the islet epigenome controls adaptive insulin secretion.

Adaptation of the islet β cell insulin-secretory response to changing insulin demand is critical for blood glucose homeostasis, yet the mechanisms underlying this adaptation are unknown. Here, we have shown that nutrient-stimulated histone acetylation plays a key role in adapting insulin secretion through regulation of genes involved in β cell nutrient sensing and metabolism. Nutrient regulation of the epigenome occurred at sites occupied by the chromatin-modifying enzyme lysine-specific demethylase 1 (Lsd1) in islets. β Cell-specific deletion of Lsd1 led to insulin hypersecretion, aberrant expression of nutrient-response genes, and histone hyperacetylation. Islets from mice adapted to chronically increased insulin demand exhibited shared epigenetic and transcriptional changes. Moreover, we found that genetic variants associated with type 2 diabetes were enriched at LSD1-bound sites in human islets, suggesting that interpretation of nutrient signals is genetically determined and clinically relevant. Overall, these studies revealed that adaptive insulin secretion involves Lsd1-mediated coupling of nutrient state to regulation of the islet epigenome.

Discovery of novel, potent, and orally bioavailable HDACs inhibitors with LSD1 inhibitory activity for the treatment of solid tumors

Histone deacetylases (HDACs) and lysine-specific demethylase 1 (LSD1) are attractive targets for epigenetic cancer therapy. There is an intimate interplay between the two enzymes. HDACs inhibitors have shown synergistic anticancer effects in combination with LSD1 inhibitors in several types of cancer. Herein, we describe the discovery of compound 5e, a highly potent HDACs inhibitor (HDAC1/2/6/8; IC50 = 2.07/4.71/2.40/107 nM) with anti-LSD1 potency (IC50 = 1.34 μM). Compound 5e exhibited marked antiproliferative activity in several cancer cell lines. 5e effectively induced mitochondrial apoptosis with G2/M phase arrest, inhibiting cell migration and invasion in MGC-803 and HCT-116 cancer cells. It also showed good liver microsomal stability and acceptable pharmacokinetic parameters in SD rats. More importantly, orally administered compound 5e demonstrated higher in vivo antitumor efficacy than SAHA in the MGC-803 (TGI = 71.5%) and HCT-116 (TGI = 57.6%) xenograft tumor models accompanied by good tolerability. This study provides a novel lead compound with dual inhibitory activity against HDACs and LSD1 to further develop epigenetic drugs for solid tumor therapy. Further optimization is needed to improve the LSD1 activity to achieve dual inhibitors with balanced potency on LSD1 and HDACs.

Long noncoding RNAs with peptide-encoding potential identified in esophageal squamous cell carcinoma: KDM4A-AS1-encoded peptide weakens cancer cell viability and migratory capacity.

Currently, the knowledge of long noncoding RNA (lncRNA)-encoded peptides is quite lacking in esophageal squamous cell carcinoma (ESCC). In this study, we simultaneously identified six lncRNA open reading frames (ORFs) with peptide-coding abilities including lysine-specific demethylase 4A antisense RNA 1 (KDM4A-AS1) ORF by combining weighted gene co-expression network analysis (WGCNA) for ESCC clinical samples, ribosome footprints, ORF prediction, mass spectrometry (MS) identification, and western blotting. KDM4A-AS1 ORF-encoded peptide reduced ESCC cell viability and migratory ability. Co-immunoprecipitation and MS analysis revealed that KDM4A-AS1-encoded peptide specifically bound with 103 proteins in ESCC cells, and enrichment analysis suggested that peptide-bound proteins were related to fatty acid metabolism and redox process. Cell and molecular experiments demonstrated that KDM4A-AS1-encoded peptide inhibited stearoyl-CoA desaturase and fatty acid synthase expression, increased reactive oxygen species level, and reduced mitochondrial membrane potential in ESCC cells. In summary, multiple lncRNAs with translation potential were simultaneously identified by combining multiple approaches in ESCC, providing novel identificationstrategies for lncRNA-encoded peptides. Moreover, lncRNA KDM4A-AS1-encoded peptide weakened ESCC cell viability and migratory capacity and functioned in fatty acid metabolism and redox process.

Oncogenic role and target properties of the lysine-specific demethylase KDM1A in chronic lymphocytic leukemia

AbstractIn chronic lymphocytic leukemia (CLL), epigenetic alterations are considered to centrally shape the transcriptional signatures that drive disease evolution and underlie its biological and clinical subsets. Characterizations of epigenetic regulators, particularly histone-modifying enzymes, are very rudimentary in CLL. In efforts to establish effectors of the CLL-associated oncogene T-cell leukemia 1A (TCL1A), we identified here the lysine-specific histone demethylase KDM1A to interact with the TCL1A protein in B cells in conjunction with an increased catalytic activity of KDM1A. We demonstrate that KDM1A is upregulated in malignant B cells. Elevated KDM1A and associated gene expression signatures correlated with aggressive disease features and adverse clinical outcomes in a large prospective CLL trial cohort. Genetic Kdm1a knockdown in Eμ-TCL1A mice reduced leukemic burden and prolonged animal survival, accompanied by upregulated p53 and proapoptotic pathways. Genetic KDM1A depletion also affected milieu components (T, stromal, and monocytic cells), resulting in significant reductions in their capacity to support CLL-cell survival and proliferation. Integrated analyses of differential global transcriptomes (RNA sequencing) and H3K4me3 marks (chromatin immunoprecipitation sequencing) in Eμ-TCL1A vs iKdm1aKD;Eμ-TCL1A mice (confirmed in human CLL) implicate KDM1A as an oncogenic transcriptional repressor in CLL which alters histone methylation patterns with pronounced effects on defined cell death and motility pathways. Finally, pharmacologic KDM1A inhibition altered H3K4/9 target methylation and revealed marked anti–B-cell leukemic synergisms. Overall, we established the pathogenic role and effector networks of KDM1A in CLL via tumor-cell intrinsic mechanisms and its impacts in cells of the microenvironment. Our data also provide rationales to further investigate therapeutic KDM1A targeting in CLL.

Computational evaluation on molecular stability and binding affinity of methyldopa against Lysine-specific demethylase 4D Enzyme through quantum chemical computations and molecular docking analysis

For the complete characterization of the molecule, quantum chemical calculations utilizing density functional theory (DFT) were performed at the B3LYP/6–311++G(d,p) level of theory. The quantum theory of atoms in molecules (QTAIM) and the reduced density gradient (RDG) analysis were implemented to investigate non-covalent interactions. The electronic environment of each atom in a molecule was explored using an electron localization function (ELF). Molecular stability of dimer structure due to intermolecular hydrogen bonds: O29-H54⋅⋅⋅O2, O1-H26⋅⋅⋅O30, and O32-H56⋅⋅⋅O4 and their corresponding stabilization energies were predicted by natural bond orbital (NBO) analysis. The energy gap and global and local reactivity parameters were determined to predict the chemical reactivity. The molecular electrostatic potential (MEP) was used to predict the electrophilic and nucleophilic sites. The frontier molecular orbitals (FMOs) were also portrayed using a density of states (DOS). Molecular docking was performed to study the interaction of the molecule with an enzyme, Lysine-specific demethylase 4D-like, a human protein.

Abstract 2673: Several LSD1 inhibitors have a potential to disturb fatty acid metabolism necessary &It glycolysis-inhibited pancreatic cancers to survive

Abstract Despite increased aerobic glycolysis is generally seen in human cancers, pancreatic ductal adenocarcinoma (PDAC) cells keep alive in glycolysis-suppressed conditions (1). Here, we sought there are potential therapeutic targets in glycolysis-suppressed PDAC cells. By screening anti-cancer metabolism compounds, we identified SP2509, a selective lysine specific demethylase 1 (LSD1) inhibitor (2). We also tested the effect of another three LSD1 inhibitors (Iadademstat, OG-L002 and T-3775440) on PDAC cells viability. The results showed that OG-L002, but not Iadademstat or T-3775440, lowered glycolysis-suppressed PDAC cells viability, like SP2509. Importantly, only knockdown of LSD1 or LSD2 failed to lower the viability of glycolysis suppressed PDAC cells. In our previous study, we proved that glucose-starvation make PDAC cells yield survival to mitochondrial oxidative phosphorylation (1). In this study, we cleared fatty acids metabolism played a crucial role for glycolysis suppressed PDAC cells survival. Then, we confirmed that SP2509 and OG-L002 can lead lipid droplets accumulation in glycolysis-suppressed conditions. In conclusion, our results showed that SP2509 and OG-L002 may involve regulatory effects on lipid droplets homeostasis, which control storage and release of fatty acids, not only by targeting LSD1 or LSD2. Moreover, SP2509 and OG-L002 significantly lowered viability of glycolysis-suppressed PDAC cells, supposing a novel therapeutic potential of approaches to override resistance of glucose-starvation in PDAC cells. 1.Shiratori R, Furuichi K, Yamaguchi M, Miyazaki N, Aoki H, Chibana H, et al. Glycolytic suppression dramatically changes the intracellular metabolic profile of multiple cancer cell lines in a mitochondrial metabolism-dependent manner. Sci Rep. 2019;9(1):18699. 2.Fiskus W, Sharma S, Shah B, Portier BP, Devaraj SGT, Liu K, et al. Highly effective combination of LSD1 (KDM1A) antagonist and pan-histone deacetylase inhibitor against human AML cells. Leukemia. 2017;31(7):1658. Citation Format: Zhiheng Zhang, Haruna Aoki, Keitaro Umezawa, Taichi Oshima, Takuya Hirao, Yuri Miura, Kousei Ito, Shigeki Aoki. Several LSD1 inhibitors have a potential to disturb fatty acid metabolism necessary &It glycolysis-inhibited pancreatic cancers to survive [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 2673.

Lysine-Specific Demethylase 1 Promises to Be a Novel Target in Cancer Drug Resistance: Therapeutic Implications.

Chemotherapy, targeted therapy, and immunotherapy are effective against most tumors, but drug resistance remains a barrier to successful treatment. Lysine-specific demethylase 1 (LSD1), a member of histone demethylation modifications, can regulate invasion, metastasis, apoptosis, and immune escape of tumor cells, which are associated with tumorigenesis and tumor progression. Recent studies suggest that LSD1 ablation regulates resensitivity of tumor cells to anticarcinogens containing immune checkpoint inhibitors (ICIs) via multiple upstream and downstream pathways. In this review, we describe the recent findings about LSD1 biology and its role in the development and progression of cancer drug resistance. Further, we summarize LSD1 inhibitors that have a reversal or resensitive effect on drug resistance and discuss the possibility of targeting LSD1 in combination with other agents to surmount resistance.

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.

Comparison of pharmacological inhibitors of lysine-specific demethylase 1 in glioblastoma stem cells reveals inhibitor-specific efficacy profiles.

Improved therapies for glioblastoma (GBM) are desperately needed and require preclinical evaluation in models that capture tumor heterogeneity and intrinsic resistance seen in patients. Epigenetic alterations have been well documented in GBM and lysine-specific demethylase 1 (LSD1/KDM1A) is amongst the chromatin modifiers implicated in stem cell maintenance, growth and differentiation. Pharmacological inhibition of LSD1 is clinically relevant, with numerous compounds in various phases of preclinical and clinical development, but an evaluation and comparison of LSD1 inhibitors in patient-derived GBM models is lacking. To assess concordance between knockdown of LSD1 and inhibition of LSD1 using a prototype inhibitor in GBM, we performed RNA-seq to identify genes and biological processes associated with inhibition. Efficacy of various LSD1 inhibitors was assessed in nine patient-derived glioblastoma stem cell (GSC) lines and an orthotopic xenograft mouse model. LSD1 inhibitors had cytotoxic and selective effects regardless of GSC radiosensitivity or molecular subtype. In vivo, LSD1 inhibition via GSK-LSD1 led to a delayed reduction in tumor burden; however, tumor regrowth occurred. Comparison of GBM lines by RNA-seq was used to identify genes that may predict resistance to LSD1 inhibitors. We identified five genes that correlate with resistance to LSD1 inhibition in treatment resistant GSCs, in GSK-LSD1 treated mice, and in GBM patients with low LSD1 expression. Collectively, the growth inhibitory effects of LSD1 inhibition across a panel of GSC models and identification of genes that may predict resistance has potential to guide future combination therapies.

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.