LSD1 Inhibitors Research Articles

LSD1 Inhibition Exerts Its Anti-Leukemic Effect By Recommissioning PU.1- and C/Ebpα-Dependent Enhancers in AML

Epigenetic regulators are recurrently mutated and aberrantly expressed in acute myeloid leukemia (AML). Targeted therapies designed to inhibit these chromatin-modifying enzymes, such as the histone demethylase lysine specific demethylase 1 (LSD1) and the histone methyltransferase DOT1L, have been developed as novel treatment modalities for these often refractory diseases. We aimed to investigate the chromatin changes induced by an irreversible inhibitor of LSD1 (GSK-LSD1) on rMLL+ leukemias. For this, we treated rMLL+ murine AML and patient-derived xenografts with the GSK-LSD1 inhibitor in vivo and assessed chromatin accessibility changes using the Assay for Transposase-Accessible Chromatin with high throughput sequencing (ATAC-seq). Treated mice exhibited a lower proportion of MLL-AF9+ leukemia cells detected in the bone marrow (BM) and signs of myeloid differentiation. Treated mice also had markedly improved survival with a median survival of 78 days vs. control mice (39 days). After 6 weeks of treatment with GSK-LSD1, mice xenotransplanted with human rMLL+ AML samples showed a significant decrease in engraftment and myeloid differentiation. Serial transplantation of murine AML revealed eradication of LSC upon GSK-LSD1 treatment of donor mice. Most importantly, LSD1 inhibition resulted in gain in chromatin accessibility in both murine and PDX-AML cells in vitro as well as in vivo . We then wanted to compare the effect of another anti-leukemic epigenetic drug, also known to induce myeloid differentiation of MLL-AF9+ cells, such as the DOT1L inhibitor EPZ4777. In contrast to GSK-LSD1, EPZ4777 treatment caused a global decrease in chromatin accessibility. The intersection of sites with dynamic accessibility upon DOT1L and LSD1 inhibition showed minimal overlap between the 2 treatments. The distribution of genomic regions with dynamic accessibility upon these drugs indicated that DOT1L inhibition affects a greater number of sites in promoter regions, whereas LSD1 inhibition affected a higher proportion of loci occupied within the intergenic and intronic regions. Furthermore, we sought to identify TF motif signatures at GSK-LSD1-induced dynamic sites. The most highly enriched motifs captured were those for myeloid TFs PU.1 and C/EBPα-β. We then performed ChIP-seq to identify sites of C/EBPα and PU.1 occupancy in the setting of GSK-LSD1 treatment. Interestingly, we observed a redistribution of PU.1 and C/EBPα occupancy upon treatment, consistent with the role of LSD1 in regulating the binding and activity of TFs. While PU.1 ChIPseq signal increased at dynamic ATAC-seq peaks in response to LSD1 inhibition, no increase in C/EBPα occupancy was observed. These dynamic sites also demonstrates GSK-LSD1-dependent co-occupancy of PU.1 and C/EBPα. We next sought to delineate whether PU.1 and C/EBPα are critical for these dynamic changes in chromatin accessibility in response to LSD1 inhibition. We performed ATAC-seq on PU.1 URE KO MLL-AF9 leukemias and C/EBPα KO MLL-AF9 leukemias treated with vehicle or GSK-LSD1. The increase in chromatin accessibility in TF-WT MLL-AF9 leukemias upon GSK-LSD1 was abrogated in both PU.1 URE KO and C/EBPα KO MLL-AF9 leukemia cells. We next assess the impact of TF-depletion on sensitivity to LSD1 inhibition. We treated PU.1 URE KO leukemias cells with GSK-LSD1 for 48 hours in vitro . These cells were more resistant to LSD1 inhibition when compared to PU.1 URE heterozygous and WT AML cells. In vivo, we reported loss of survival benefit upon GSK-LSD1 treatment in PU.1 URE KO MLL-AF9+ mice vs. WT mice. Similarly, C/EBPα KO leukemia cells were more resistant to LSD1 inhibition when compared to WT leukemia cells. After 2 weeks of treatment in vivo, C/EBPα KO leukemic mice also showed no decrease in MLL-AF9 allele burden. Taken together, our comparative investigations into the chromatin-modifying effects of two distinct epigenetic therapies targeting either LSD1 or DOT1L is illustrative of the concept that multiple paths toward myeloid differentiation can potentially be engaged to achieve therapeutic efficacy in AML. Our findings reveal that pharmacologic inhibition of LSD1 depends on balanced expression of the TFs PU.1 and C/EBPα, which can be used as predictors of response in AML aberrantly expressing these factors. DisclosuresMohammad:GSK: Employment. Ye:Incyte: Employment. Will:Novartis Pharmaceuticals: Consultancy, Research Funding. Steidl:Celgene: Consultancy; Bayer Healthcare: Consultancy; Aileron Therapeutics: Consultancy, Research Funding; Novartis: Research Funding; GlaxoSmithKline: Research Funding. Kruger:GSK: Employment. Armstrong:Imago: Consultancy; Cyteir: Consultancy; C4 Therapeutics: Consultancy; Epizyme: Consultancy.

Oral Administration of the LSD1 Inhibitor OG-S1335 Increases Fetal Hemoglobin in Humanized Transgenic Sickle Cell Disease Mice and in Baboons

Increased levels of fetal hemoglobin (HbF) lessen the severity of symptoms and increase the life span of patients with sickle cell disease (SCD). New and more effective strategies to increase HbF are needed because the current standard of care, hydroxyurea, is not effective in a significant proportion of patients. Although BM transplantation, gene therapy, and gene editing technologies offer the promise of a cure, it is unlikely that these methodologies can be implemented to treat the large numbers of patients projected worldwide, including those in developing nations that could most easily be treated with an effective, orally administered drug therapy that increased HbF. The lysine-specific demethylase 1A (KDM1A, also referred to as LSD1), is a component of co-repressor complexes that repress γ-globin gene expression and a therapeutic target for HbF reactivation (Shi et al, Nat Med 19:291, 2013). Our laboratory has shown that subcutaneous administration of RN-1, a pharmacological inhibitor of LSD1, increased γ-globin expression in SCD transgenic mice (Rivers et al, Exp Hematol 43:546, 2015; Cui et al, Blood 126:386, 2015) and in baboons (Rivers et al, Haematol 101:688, 2016), widely acknowledged as the best animal model to test the activity of HbF-inducing drugs due to conservation of structure and developmental regulation of the β-globin locus among simian primates. The objective of this investigation was to test whether oral administration of a new LSD1 inhibitor, OG-S1335, could increase HbF in SCD transgenic mice and baboons. Oral administration of varying doses (0.25, 0.5, 1.0mg/kg/d; 4d) of OG-S1335 to SCD mice (3 mice per dose) increased γ-globin expression and the percentage of circulating erythrocytes and reticulocytes positive for HbF (F cells and F retics; Table 1). In baboons, dose escalation was initially performed by administering the drug to a single animal in cycles of 3 days, followed by 1-2 weeks of observation to measure effects on blood parameters. Increased levels of F retics were initially observed at a dose of 100μg/kg/d and subsequently 4 additional baboons were treated with OG-S1335 (by oral gavage) at a dose of either 100 or 200μg/kg/d (7 separate experiments in total). The data presented are in the mean+SD format, with “post-dose” indicating the time point when the highest variation from baseline levels was reported. Increased F retics (pre=8.4+8.9%, post=43.0+27.9%; p<0.01), γ-globin chain synthesis (pre=0.05+0.05 γ/γ+β, post=0.28+0.18 γ/γ+β; p<0.01), and γ-globin mRNA (pre=0.02 +0.02 γ/γ+β, post=0.20+0.11 γ/γ+β) were observed with maximal levels achieved 7 days after administration of the first dose (d8). A decrease in the absolute number of reticulocytes (pre=72.8+17.8 X104/μL, post=35.4+17.8 X 104/μL; p<0.05; nadir on d6-8), neutrophils (pre=4634+994/μL, post=2655+1257/μL; p<0.02; nadir on d13-17), and platelets (pre=406+86 X103/μL, post=168+98 X103/μL; p<0.01; nadir on d8) was also reported, together with increased monocytes (pre=200+68/μL, post=541+213/μL; p<0.01; peak on d10-15). A negative correlation between decreased platelet counts and F retics (r= -0.55), γ-globin chain synthesis (r= -0.92), and γ-globin mRNA (r= -0.80) suggests that variability in drug exposure in addition to possible genetic factors regulating γ-globin expression may be responsible for the observed variability between animals. Additional experiments were performed in two phlebotomized anemic baboons to test the effect of the drug in a model with increased relevance to sickle cell disease. OG-S1335 (100μg/kg/d; 3d) induced high levels of F retics (PA8695, pre-dose=24%, post=66.8%; PA8698: pre-dose=13%, post=93.6%), γ-globin chain synthesis (PA8695: pre-dose=0.07 γ/γ+β, post=0.20 γ/γ+β; PA8698: pre-dose=0.02 γ/γ+β, post=0.44 γ/γ+β) and γ-globin mRNA (PA8695: pre-dose=0.06 γ/γ+β, post=0.18 γ/γ+β; PA8698: pre-dose=0.03 γ/γ+β, post=0.33 γ/γ+β) in each animal. We conclude that oral administration of the LSD1 inhibitor OG-S1335 significantly increased F retics, γ-globin chain synthesis, and γ-globin mRNA in SCD mice and in baboons, supporting further efforts toward the development of this drug for SCD therapy. [Display omitted] DisclosuresCiceri:Oryzon Genomics SA: Employment. Cavalcanti:Oryzon Genomics SA: Employment.

Myofibroblast Deficiency of LSD1 Alleviates TAC-Induced Heart Failure.

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SP2509, an inhibitor of LSD1, exerts potential antitumor effects by targeting the JAK/STAT3 signaling.

Hyperactivation of Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling promotes tumorigenesis and cancer progression. STAT3 participates in the essential processes of cell proliferation, survival, and differentiation in many types of tumors. In the present study, SP2509 was identified as a potent inhibitor of the JAK/STAT3 signaling pathway by high-throughput drug screening based on a STAT3-driven luciferase expression system. Our results indicated that SP2509 inhibits constitutive STAT3 activation and the expression of STAT3-driven downstream genes. Bcl-xL, c-Myc, and Cyclin D1 were downregulated after treatment with SP2509. In addition, SP2509 specifically inhibits JAK activity, which could cause cell cycle arrest, inhibit cell growth, and induce apoptosis of various cancer cells. These results confirmed that SP2509 inhibits tumor progression by suppressing the expression of JAK/STAT3 signaling and STAT3-related downstream genes. Moreover, we demonstrated that SP2509 inhibits tumor growth in vivo and induces cell death in vitro. SP2509-mediated inhibition of STAT3 phosphorylation is dependent on its original target lysine-specific demethylase 1 in cancer cells. In summary, our results indicate that SP2509 is a novel inhibitor of JAK/STAT3 signaling.

Combined epigenetic and metabolic treatments overcome differentiation blockade in acute myeloid leukemia

SummaryA hallmark of acute myeloid leukemia (AML) is the inability of self-renewing malignant cells to mature into a non-dividing terminally differentiated state. This differentiation block has been linked to dysregulation of multiple cellular processes, including transcriptional, chromatin, and metabolic regulation. The transcription factor HOXA9 and the histone demethylase LSD1 are examples of such regulators that promote differentiation blockade in AML. To identify metabolic targets that interact with LSD1 inhibition to promote myeloid maturation, we screened a small molecule library to identify druggable substrates. We found that differentiation caused by LSD1 inhibition is enhanced by combined perturbation of purine nucleotide salvage and de novo lipogenesis pathways, and identified multiple lines of evidence to support the specificity of these pathways and suggest a potential basis of how perturbation of these pathways may interact synergistically to promote myeloid differentiation. In sum, these findings suggest potential drug combination strategies in the treatment of AML.

LSD1 and Aberrant DNA Methylation Mediate Persistence of Enteroendocrine Progenitors That Support BRAF-Mutant Colorectal Cancer.

Despite the connection of secretory cells, including goblet and enteroendocrine (EEC) cells, to distinct mucus-containing colorectal cancer histologic subtypes, their role in colorectal cancer progression has been underexplored. Here, our analysis of The Cancer Genome Atlas (TCGA) and single-cell RNA-sequencing data demonstrates that EEC progenitor cells are enriched in BRAF-mutant colorectal cancer patient tumors, cell lines, and patient-derived organoids. In BRAF-mutant colorectal cancer, EEC progenitors were blocked from differentiating further by DNA methylation and silencing of NEUROD1, a key gene required for differentiation of intermediate EECs. Mechanistically, secretory cells and the factors they secrete, such as trefoil factor 3, promoted colony formation and activation of cell survival pathways in the entire cell population. Lysine-specific demethylase 1 (LSD1) was identified as a critical regulator of secretory cell specification in vitro and in a colon orthotopic xenograft model, where LSD1 loss blocks formation of EEC progenitors and reduces tumor growth and metastasis. These findings reveal an important role for EEC progenitors in supporting colorectal cancer. SIGNIFICANCE: This study establishes enteroendocrine progenitors as a targetable population that promotes BRAF-mutant colorectal cancer and can be blocked by LSD1 inhibition to suppress tumor growth.

Targeting novel LSD1-dependent ACE2 demethylation domains inhibits SARS-CoV-2 replication

Treatment options for COVID-19 remain limited, especially during the early or asymptomatic phase. Here, we report a novel SARS-CoV-2 viral replication mechanism mediated by interactions between ACE2 and the epigenetic eraser enzyme LSD1, and its interplay with the nuclear shuttling importin pathway. Recent studies have shown a critical role for the importin pathway in SARS-CoV-2 infection, and many RNA viruses hijack this axis to re-direct host cell transcription. LSD1 colocalized with ACE2 at the cell surface to maintain demethylated SARS-CoV-2 spike receptor-binding domain lysine 31 to promote virus–ACE2 interactions. Two newly developed peptide inhibitors competitively inhibited virus–ACE2 interactions, and demethylase access to significantly inhibit viral replication. Similar to some other predominantly plasma membrane proteins, ACE2 had a novel nuclear function: its cytoplasmic domain harbors a nuclear shuttling domain, which when demethylated by LSD1 promoted importin-α-dependent nuclear ACE2 entry following infection to regulate active transcription. A novel, cell permeable ACE2 peptide inhibitor prevented ACE2 nuclear entry, significantly inhibiting viral replication in SARS-CoV-2-infected cell lines, outperforming other LSD1 inhibitors. These data raise the prospect of post-exposure prophylaxis for SARS-CoV-2, either through repurposed LSD1 inhibitors or new, nuclear-specific ACE2 inhibitors.

Phase 1 trial of seclidemstat (SP-2577) in patients with relapsed/refractory Ewing sarcoma.

11514 Background: Ewing sarcoma (ES), a rare bone and soft tissue sarcoma mainly of adolescents and young adults, is characterized by a chromosomal translocation resulting in a fusion oncoprotein. Lysine specific demethylase 1 (LSD1) has been shown to associate with the fusion oncoprotein and promote oncogenic transcriptional activity making LSD1 an attractive target for ES treatment. Seclidemstat is a novel, selective, reversible oral LSD1 inhibitor capable of inhibiting both LSD1’s catalytic and scaffolding functions. This is the first report of an LSD1 inhibitor in a Phase 1 trial focused exclusively on ES. Methods: SALA-002-EW16 is a Phase 1 trial of single agent seclidemstat in patients (pts) with relapsed or refractory (R/R) ES. This report describes the completed monotherapy dose escalation. Pts &gt; 12 years received oral SP-2577 twice daily in 28-day cycles under fasting conditions at the assigned dose level. The primary objective was safety and tolerability. Secondary objectives include to determine maximum-tolerated dose (MTD), recommended Phase 2 dose (RP2D), preliminary efficacy, pharmacokinetics, and pharmacodynamics. Results: As of December 30, 2020, 27 pts with R/R ES were enrolled. Pts received escalating doses of SP-2577 at 75 (n = 1), 150 (n = 2), 300 (n = 4), 600 (n = 6), 900 (n = 8), or 1200 mg PO BID (n = 6). The median age was 25 years (range 15–68), 63% were male, and pts had received a median of 3 (range 2–12) prior systemic therapies. There were no treatment-related deaths. The most common ( &gt; 5%) grade 3 treatment-related adverse events (TRAEs) were vomiting (15%), abdominal pain (11%), and hypokalemia (11%). One pt (4%) with grade 3 pancreatitis reported a grade 4 AE of elevated lipase. All remaining grade 3 TRAEs, including hematological TRAEs, were reported in only one pt each. Four pts discontinued study for an AE (weight loss, pancreatitis, vomiting, abdominal pain). Three pts had a dose reduction. The first cycle dose-limiting toxicities were gastrointestinal-related AEs observed in 2 pts at 1200 mg BID. Thus, the MTD/RP2D was established as 900 mg BID. Peak plasma concentrations occurred at a median of 4 hours (h) post-dose and median terminal half-life was 6 h; exposure was dose proportional through 900 mg BID. One pt at 600 mg BID achieved a reduction in target lesions starting at end of C2 with further target lesion tumor shrinkage through end of C4 and C6 (maximum 76% tumor shrinkage) with coincident new non-target lesion appearance at end of C2. Of pts evaluable for response at the end of C2 (12 pts), two additional pts (16.7%) at 600 mg BID and 900 mg BID had overall stable disease. Conclusions: Seclidemstat has a manageable safety profile with proof-of-concept preliminary activity in heavily pretreated pts with relapsed/refractory ES. These data support the planned Phase 2 expansion of seclidemstat as single agent and in combination with chemotherapy in ES and other sarcomas that share similar translocations. Clinical trial information: NCT03600649.

Phase 1 expansion trial of the LSD1 inhibitor seclidemstat (SP-2577) with and without topotecan and cyclophosphamide (TC) in patients (pts) with relapsed or refractory Ewing sarcoma (ES) and select sarcomas.

TPS11577 Background: Several sarcomas possess chromosomal translocations in FET family members ( FUS, EWSR1, and TAF15) responsible for cancer development. Sarcomas caused by FET family gene rearrangements include ES, desmoplastic round cell small tumors (DSRCT), myxoid liposarcoma (ML), and several others. Lysine specific demethylase 1 (LSD1) is a critical protein for sarcoma development and progression through its colocalization and/or association with several FET family oncogenic transcription factors. This suggests that pharmacologic inhibition of LSD1 may be a therapeutic strategy. Seclidemstat (SP-2577, Salarius Pharmaceuticals) is an oral, first-in-class, small molecule with reversible, noncompetitive inhibition of LSD1 (IC50: 25–50 nM). In vitro and in vivo data demonstrate seclidemstat, or analogs, modulate EWS/ETS transcriptional activity, down-regulating oncogene expression and up-regulating tumor-suppressor gene expression, leading to significant tumor growth inhibition in ES mouse xenograft studies. Seclidemstat has shown in in vitro ES cell lines near additivity efficacy when added to TC. In in vitro studies of other FET-translocated sarcomas, including ML (FUS/DDIT3 fusion) and clear cell sarcoma (EWS/ATF1 fusion), seclidemstat showed anti-proliferative activity. In an ongoing Phase 1 trial investigating single agent seclidemstat in advanced solid tumors (NCT03895684), three pts with metastatic FET-translocated sarcomas had a median progression-free survival of 5.7 months (range: 4.3–7.2) with a best response of stable disease despite having a median of 5 (range: 1–7) prior therapies. Methods: This dose expansion Phase 1 study (NCT03600649) assesses seclidemstat at 900 mg PO BID, the recommended Phase 2 dose, in two expansion cohorts: a single agent expansion in select sarcoma pts (n = 30) and a safety lead-in dose escalation and expansion (n = 24) of seclidemstat combined with TC in pts with ES. Pts must be ≥12 years old, have ECOG performance status of 0 or 1, with a life expectancy &gt; 4 months. In the select sarcoma cohort, pts must have ML (n = 15) or other sarcomas with FET family translocations (n = 15) including DSRCT. One to 3 prior lines of therapy are allowed. In the ES combination cohort, up to 2 lines of prior therapy are allowed. Primary objective is safety/tolerability and secondary objective is efficacy. The trial is currently recruiting across 8 locations in the United States. Clinical trial information: NCT03600649.

Preliminary efficacy from an ongoing phase 1 dose escalation study of seclidemstat (SP-2577) in patients (pts) with advanced solid tumors (AST).

3073 Background: Lysine-specific demethylase 1 (LSD1) is an epigenetic enzyme that is aberrantly expressed in many solid tumors. High levels of LSD1 expression are often correlated with poor patient prognosis due to LSD1’s role in cancer cell proliferation, metastasis, and chemoresistance. Seclidemstat is a novel, selective, reversible and oral LSD1 inhibitor capable of inhibiting both LSD1’s catalytic and scaffolding functions. We report preliminary efficacy in AST from an ongoing phase 1 trial. Methods: SALA-003-AC19 (NCT03895684) is a phase 1 trial of single agent SP-2577 in pts with AST. All pts had progressive disease (PD) at time of study entry. Pts received oral SP-2577 twice a day under fasting condition, in 28-day cycles (C). The primary objective is safety and tolerability. Secondary objectives are to determine maximum-tolerated dose, preliminary efficacy, pharmacokinetics, and pharmacodynamics. Results: As of December 30, 2020, 19 pts with AST (10 sarcoma, 2 prostate, 2 ovarian, 2 pancreatic, 1 renal, 1 cervical, 1 breast) were enrolled. Pts received escalating doses of SP-2577 from 150 to 600 mg BID and the dose escalation is ongoing. The median age was 63 years (range, 21–79). 42% were male, and pts had received a median of 4 (range, 1–8) prior systemic therapies. The most common (&gt;5%) grade 3 treatment-related adverse events were GI related including diarrhea (5.3%) and abdominal pain (5.3%). No grade 4 events were reported and there were no treatment-related deaths. Safety data will be presented after completion of phase 1. Three pts had at least one dose reduction. Among the 13 pts who were evaluable for response at end of C2, 7 pts (54%) had best response of stable disease (SD) with median time to progression (TTP) of 4.3 months (range, 2.1–11.5). Four of the 7 pts had genetic abnormalities that may demonstrate increased sensitization to SP-2577 according to preclinical studies. Characteristics of 7 pts with SD at C2 and beyond are shown in the table. Conclusions: Seclidemstat has shown activity among advanced sarcoma pts with a manageable safety profile. The dose escalation is ongoing and preliminary clinical data supports further exploration in FET-translocated sarcoma as single agent and in combination therapy. Clinical trial information: NCT03895684. [Table: see text]

Superior efficacy of co-targeting GFI1/KDM1A and BRD4 against AML and post-MPN secondary AML cells

There is an unmet need to overcome nongenetic therapy-resistance to improve outcomes in AML, especially post-myeloproliferative neoplasm (MPN) secondary (s) AML. Studies presented describe effects of genetic knockout, degradation or small molecule targeted-inhibition of GFI1/LSD1 on active enhancers, altering gene-expressions and inducing differentiation and lethality in AML and (MPN) sAML cells. A protein domain-focused CRISPR screen in LSD1 (KDM1A) inhibitor (i) treated AML cells, identified BRD4, MOZ, HDAC3 and DOT1L among the codependencies. Our findings demonstrate that co-targeting LSD1 and one of these co-dependencies exerted synergistic in vitro lethality in AML and post-MPN sAML cells. Co-treatment with LSD1i and the JAKi ruxolitinib was also synergistically lethal against post-MPN sAML cells. LSD1i pre-treatment induced GFI1, PU.1 and CEBPα but depleted c-Myc, overcoming nongenetic resistance to ruxolitinib, or to BETi in post-MPN sAML cells. Co-treatment with LSD1i and BETi or ruxolitinib exerted superior in vivo efficacy against post-MPN sAML cells. These findings highlight LSD1i-based combinations that merit testing for clinical efficacy, especially to overcome nongenetic therapy-resistance in AML and post-MPN sAML.

LSD1 defines erythroleukemia metabolism by controlling the lineage-specific transcription factors GATA1 and C/EBPα

Acute myeloid leukemia (AML) is a heterogenous malignancy characterized by distinct lineage subtypes and various genetic/epigenetic alterations. As with other neoplasms, AML cells have well-known aerobic glycolysis, but metabolic variations depending on cellular lineages also exist. Lysine-specific demethylase-1 (LSD1) has been reported to be crucial for human leukemogenesis, which is currently one of the emerging therapeutic targets. However, metabolic roles of LSD1 and lineage-dependent factors remain to be elucidated in AML cells. Here, we show that LSD1 directs a hematopoietic lineage-specific metabolic program in AML subtypes. Erythroid leukemia (EL) cells particularly showed activated glycolysis and high expression of LSD1 in both AML cell lines and clinical samples. Transcriptome, chromatin immunoprecipitation–sequencing, and metabolomic analyses revealed that LSD1 was essential not only for glycolysis but also for heme synthesis, the most characteristic metabolic pathway of erythroid origin. Notably, LSD1 stabilized the erythroid transcription factor GATA1, which directly enhanced the expression of glycolysis and heme synthesis genes. In contrast, LSD1 epigenetically downregulated the granulo-monocytic transcription factor C/EBPα. Thus, the use of LSD1 knockdown or chemical inhibitor dominated C/EBPα instead of GATA1 in EL cells, resulting in metabolic shifts and growth arrest. Furthermore, GATA1 suppressed the gene encoding C/EBPα that then acted as a repressor of GATA1 target genes. Collectively, we conclude that LSD1 shapes metabolic phenotypes in EL cells by balancing these lineage-specific transcription factors and that LSD1 inhibitors pharmacologically cause lineage-dependent metabolic remodeling.

Design, synthesis, and biological evaluation of novel dual inhibitors targeting lysine specific demethylase 1 (LSD1) and histone deacetylases (HDAC) for treatment of gastric cancer.

LSD1 and HDAC are physical and functional related to each other in various human cancers and simultaneous pharmacological inhibition of LSD1 and HDAC exerts synergistic anti-cancer effects. In this work, a series of novel LSD1/HDAC bifunctional inhibitors with a styrylpyridine skeleton were designed and synthesized based on our previously reported LSD1 inhibitors. The representative compounds 5d and 5m showed potent activity against LSD1 and HDAC at both molecular and cellular level and displayed high selectivity against MAO-A/B. Moreover, compounds 5d and 5m demonstrated potent antiproliferative activities against MGC-803 and HCT-116 cancer cell lines. Notably, compound 5m showed superior invitro anticancer potency against a panel of gastric cancer cell lines than ORY-1001 and SP-2509 with IC50 values ranging from 0.23 to 1.56μM. Compounds 5d and 5m significantly modulated the expression of Bcl-2, Bax, Vimentin, ZO-1 and E-cadherin, induced apoptosis, reduced colony formation and suppressed migration in MGC-803 cancer cells. In addition, preliminary absorption, distribution, metabolism, excretion (ADME) studies revealed that compounds 5d and 5m showed acceptable metabolic stability in human liver microsomes with minimal inhibition of cytochrome P450s (CYPs). Those results indicated that compound 5m could be a promising lead compound for further development as a therapeutic agent in gastric cancers via LSD1 and HDAC dual inhibition.

Design, synthesis and biological evaluation of novel benzofuran derivatives as potent LSD1 inhibitors.

Lysine-specific demethylase 1 (LSD1) is a FAD-dependent enzyme, which has been proposed as a promising target for therapeutic cancer. Herein, a series of benzofuran derivatives were designed, synthesized and biochemical evaluated as novel LSD1 inhibitors based on scaffold hopping and conformational restriction strategy. Most of the compounds potently suppressed the enzymatic activities of LSD1 and potently inhibited tumor cells proliferation. In particular, the representative compound 17i exhibited excellent LSD1 inhibition at the molecular levels with IC50=0.065μM, as well as anti-proliferation against MCF-7, MGC-803, H460, A549 and THP-1 tumor cells with IC50 values of 2.90±0.32, 5.85±0.35, 2.06±0.27, 5.74±1.03 and 6.15±0.49μM, respectively. The binding modes of these compounds were rationalized by molecular docking. Meanwhile, a preliminary druggability evaluation showed that compound 17i displayed favorable liver microsomal stability and weak inhibitory activity against CYPs at 10μM. Remarkably, H460 xenograft tumors studies revealed that 17i demonstrated robust invivo antitumor efficacy without significant side effects. All the results demonstrated that compound 17i could represent a promising lead for further development.

Lysine-specific demethylase 1 inhibition enhances autophagy and attenuates early-stage post-spinal cord injury apoptosis

Neuron death in spinal cords is caused primarily by apoptosis after spinal cord injury (SCI). Autophagy can act as a cellular response to maintain neuron homeostasis that can reduce apoptosis. Although more studies have shown that an epigenetic enzyme called Lysine-specific demethylase 1 (LSD1) can negatively regulate autophagy during cancer research, existing research does not focus on impacts related to LSD1 in nerve injury diseases. This study was designed to determine whether inhibiting LSD1 could enhance autophagy against apoptosis and provide effective neuroprotection in vitro and vivo after SCI. The results showed that LSD1 inhibition treatment significantly reduced spinal cord damage in SCI rat models and was characterized by upregulated autophagy and downregulated apoptosis. Further research demonstrated that using both pharmacological inhibition and gene knockdown could enhance autophagy and reduce apoptosis for in vitro simulation of SCI-caused damage models. Additionally, 3-methyladenine (3-MA) could partially eliminate the effect of autophagy enhancement and apoptosis suppression. These findings demonstrated that LSD1 inhibition could protect against SCI by activating autophagy and hindering apoptosis, suggesting a potential candidate for SCI therapy.

Epigenetic Remodeling Hydrogel Patches for Multidrug-Resistant Triple-Negative Breast Cancer.

The induced expansion of tumor-initiating cells (T-ICs) upon repeated exposure of tumors to chemotherapeutic drugs forms a major cause for chemoresistance and cancer metastasis. Here, a tumor-microenvironment-responsive hydrogel patch is designed to modulate the plasticity of T-ICs in triple-negative breast cancer (TNBC), which is insensitive to hormone- and HER2-targeting. The on-site formation of the hydrogel network patches tumors in a chemoresistant TNBC murine model and senses intratumoral reactive oxygen species for linker cleavage and payload release. Patch-mediated inhibition of the histone demethylase lysine-specific demethylase 1 (LSD1) epigenetically regulates the switch of T-ICs from self-renewal to differentiation, rehabilitating their chemosensitivity. Moreover, the hydrogel patch enhances tumor immunogenicity and increases T-cell infiltration via epigenetic activation of innate immunity. A single-dose of the hydrogel patch harboring LSD1 inhibitor and chemotherapy agent efficiently suppresses tumor growth, postsurgical relapse, and metastasis. The superior efficacy against multidrug resistance further reveals the broad applicability of epigenetic remodeling hydrogel patches.

Inhibition of histone demethylases as an approach to restore deficient DOT1L activity in osteoarthritis

Purpose: Osteoarthritis (OA), the most common chronic joint disease worldwide, is characterized by progressive damage to the articular cartilage, increased joint-associated bone remodelling, and synovial inflammation. Current OA treatments are limited to pain relief, physiotherapy, or replacement surgeries in severe cases, yet disease-modifying drugs are lacking. A genome wide association study (GWAS) revealed a genetic association between polymorphisms in the DOT1L gene and OA. The Disruptor of telomeric silencing 1-like (DOT1L) gene encodes a histone methyltransferase that methylates Lysine 79 of Histone H3 (H3K79). We identified DOT1L as a key protector of cartilage homeostasis, using both human articular chondrocytes and different Dot1l genetic mouse models. Furthermore, we reported that DOT1L activity, indicated by the levels of H3K79 methylation (H3K79me), is reduced in OA as compared to non-OA cartilage. Therefore, maintaining H3K79me seems to be critical to preserve joint health and prevent the development or progression of OA. Here, we hypothesized that H3K79me could be restored or maintained by inhibiting demethylation at the H3K79 site, via targeting specific histone demethylases. There are two main families of histone demethylases: the lysine-specific demethylases (LSD) and Jumonji C (JmjC) demethylases. The LSD family contains two members: LSD1 and LSD2. The JmjC family is further classified into 6 subfamilies: from KDM2 to KDM7. In this new study, we aimed to investigate which histone demethylases are responsible for H3K79 demethylation and whether their specific targeting can lead to protective effects in OA. Methods: We determined the baseline mRNA expression of 28 reported histone demethylases in primary human articular chondrocytes (hACs) from non-OA patients, using real-time qPCR. We also determined the changes in histone demethylase expression in vitro upon OA-mimicking stimuli, namely proinflammatory cytokine IL-1β and the Wnt signalling activator CHIR99021. To interrogate the role of JmjC demethylase family in H3K79me, human articular chondrocyte C28/I2 cells were treated with the JmjC pan inhibitor JIB-04. In parallel, the LSD family members LSD1 and LSD2 were pharmacologically inhibited using LSD1 inhibitor II or silenced using siRNA against LSD2, respectively. The role of the different JmjC subfamilies on H3K79me was studied using selective pharmacological inhibitors. We assessed the levels of H3K79me by Western blot and immunofluorescence analysis. The translational impact of individual JmjC demethylase targeting was evaluated using a siRNA knockdown strategy in hACs from OA patients. In this setup, gene expression of healthy cartilage markers Collagen2a1 (COL2A1) and Aggrecan, as well as of catabolic markers MMP13 and ADAMTS5, was assessed using real-time qPCR. Results: The baseline expression of histone demethylases was mapped in hACs, showing differences in absolute expression among the different enzymes. Treatment with IL1-β resulted in an increase in KDM6B and KDM7A mRNA expression. Conversely, Wnt signalling activation by CHIR99021 led to a downregulation in most of histone demethylases’ mRNA expression. Interestingly, pharmacological inhibition of the JmjC family using JIB-04 resulted in increased H3K79me levels. However, the blockade of LSD family members did not lead to H3K79me changes in articular chondrocytes. Moreover, inhibition of all JmjC demethylase subfamilies increased H3K79me levels. Individual silencing of specific JmjC demethylases increased the expression of chondrocyte healthy markers while reducing the expression of catabolic markers. Conclusions: The discovery approach performed in this study unravels that histone demethylases from the JmjC family control H3K79me in the articular cartilage. Silencing of individual demethylases from this family exerted protective effects in the articular chondrocyte. Therefore, targeting specific histone demethylases could restore the loss of DOT1L activity in OA and lead to therapeutic benefits in this chronic disease.

50P A phase Ib study of CC-90011, a potent, reversible, oral LSD1 inhibitor, plus etoposide and cisplatin (EP) or carboplatin (EC) in patients (Pts) with first-line (1L) extensive-stage (ES) small cell lung cancer (SCLC): Updated results

50P A phase Ib study of CC-90011, a potent, reversible, oral LSD1 inhibitor, plus etoposide and cisplatin (EP) or carboplatin (EC) in patients (Pts) with first-line (1L) extensive-stage (ES) small cell lung cancer (SCLC): Updated results

Discovery of new tranylcypromine derivatives as highly potent LSD1 inhibitors

Tranylcypromine (TCP)-based structural modifications lead to the discovery of new LSD1 inhibitors, of which compounds 26b and 29b effectively inhibit LSD1 with the IC50 values of 17 and 11 nM, respectively and also show good selectivity over MAO-B. Mechanistic studies showed that compound 29b concentration-dependently induced H3K4me1/2 accumulation in LSD1 overexpressed MGC-803 cells and also inhibited metastasis of MGC-803 cells. Collectively, both compounds could be promising lead compounds for further investigation.

LSD1 as a Biomarker and the Outcome of Its Inhibitors in the Clinical Trial: The Therapy Opportunity in Tumor.

Tumors are the foremost cause of death worldwide. As a result of that, there has been a significant enhancement in the investigation, treatment methods, and good maintenance practices on cancer. However, the sensitivity and specificity of a lot of tumor biomarkers are not adequate. Hence, it is of inordinate significance to ascertain novel biomarkers to forecast the prognosis and therapy targets for tumors. This review characterized LSD1 as a biomarker in different tumors. LSD1 inhibitors in clinical trials were also discussed. The recent pattern advocates that LSD1 is engaged at sauce chromatin zones linking with complexes of multi-protein having an exact DNA-binding transcription factor, establishing LSD1 as a favorable epigenetic target, and also gives a large selection of therapeutic targets to treat different tumors. This review sturdily backing the oncogenic probable of LSD1 in different tumors indicated that LSD1 levels can be used to monitor and identify different tumors and can be a useful biomarker of progression and fair diagnosis in tumor patients. The clinical trials showed that inhibitors of LSD1 have growing evidence of clinical efficacy which is very encouraging and promising. However, for some of the inhibitors such as GSK2879552, though selective, potent, and effective, its disease control was poor as the rate of adverse events (AEs) was high in tumor patients causing clinical trial termination, and continuation could not be supported by the risk-benefit profile. Therefore, we propose that, to attain excellent clinical results of inhibitors of LSD1, much attention is required in designing appropriate dosing regimens, developing in-depth in vitro/in vivo mechanistic works of LSD1 inhibitors, and developing inhibitors of LSD1 that are reversible, safe, potent, and selective which may offer safer profiles.