Reversible Lysine Specific Demethylase 1 Inhibitors Research Articles

Optimization and Biological Evaluation of Novel 1H-Pyrrolo[2,3-c]pyridin Derivatives as Potent and Reversible Lysine Specific Demethylase 1 Inhibitors for the Treatment of Acute Myelogenous Leukemia.

Lysine-specific demethylase 1 (LSD1) plays a vital role in the epigenetic regulation of various cancers, making it a promising therapeutic target for anticancer treatments. Herein, we designed and synthesized a novel series of 1H-pyrrolo[2,3-c]pyridin derivatives as potent LSD1 inhibitors. A detailed structure-activity relationship exploration was carried out to discover multiple derivatives with nanomolar enzymatic IC50 values. Further biological evaluation demonstrated that these compounds acted as selective and reversible LSD1 inhibitors. The representative compounds exhibited highly potent antiproliferative activity against both AML (MV4-11 and Kasumi-1) and SCLC (NCI-H526) cell lines. Additionally, they effectively activated CD86 mRNA expression in MV4-11 cells and induced differentiation of AML cell lines. Notably, the most promising compound 23e showed a favorable oral PK profile and effectively suppressed the tumor growth in an AML xenograft model. Overall, our medicinal chemistry efforts provide compound 23e as a lead compound for developing LSD1 inhibitors for the treatment of AML and other advanced malignancies.

Discovery of Pyrrolo[2,3-c]pyridines as Potent and Reversible LSD1 Inhibitors.

Lysine specific demethylase 1 (LSD1) acts as an epigenetic eraser by specifically demethylating mono- and histone 3 lysine 4 (H3K4) and H3 lysine 9 (H3K9) residues. LSD1 has been pursued as a promising therapeutic target for the treatment of human cancer, and a number of LSD1 inhibitors have been advanced into clinical development. In the present study, we describe our discovery of pyrrolo[2,3-c]pyridines as a new class of highly potent and reversible LSD1 inhibitors, designed on the basis of a previously reported LSD1 inhibitor GSK-354. Among them, 46 shows an IC50 value of 3.1 nM in inhibition of LSD1 enzymatic activity and inhibits cell growth with IC50 values of 0.6 nM in the MV4;11 acute leukemia cell line and 1.1 nM in the H1417 small-cell lung cancer cell line. Compound 46 (LSD1-UM-109) is a novel, highly potent, and reversible LSD1 inhibitor and serves as a promising lead compound for further optimization.

LSD1-Based Reversible Inhibitors Virtual Screening and Binding Mechanism Computational Study.

As one of the crucial targets of epigenetics, histone lysine-specific demethylase 1 (LSD1) is significant in the occurrence and development of various tumors. Although several irreversible covalent LSD1 inhibitors have entered clinical trials, the large size and polarity of the FAD-binding pocket and undesired toxicity have focused interest on developing reversible LSD1 inhibitors. In this study, targeting the substrate-binding pocket of LSD1, structure-based and ligand-based virtual screenings were adopted to expand the potential novel structures with molecular docking and pharmacophore model strategies, respectively. Through drug-likeness evaluation, ADMET screening, molecular dynamics simulations, and binding free energy screening, we screened out one and four hit compounds from the databases of 2,029,554 compounds, respectively. Generally, these hit compounds can be divided into two categories, amide (Lig2 and Comp2) and 1,2,4-triazolo-4,3-α-quinazoline (Comp3, Comp4, Comp7). Among them, Comp4 exhibits the strongest binding affinity. Finally, the binding mechanisms of the hit compounds were further calculated in detail by the residue free energy decomposition. It was found that van der Waals interactions contribute most to the binding, and FAD is also helpful in stabilizing the binding and avoiding off-target effects. We believe this work not only provides a solid theoretical foundation for the design of LSD1 substrate reversible inhibitors, but also expands the diversity of parent nucleus, offering new insights for synthetic chemists.

Abstract 1048: Synergistic drug combination of LSD1 and ABL2 inhibitors for Ewing's sarcoma identified in an arrayed CRISPR screen approach

Abstract Background: Ewing's Sarcoma (ES) is a rare and aggressive pediatric bone tumor that is driven by a chromosomal translocation and fusion between EWS and FLI1 genes resulting in a chimeric protein. Nearly 85% of ES cases express the EWS/FLI protein which functions as a transcription factor and drives oncogenesis. EWS/FLI interacts with lysine-specific demethylase 1 (LSD1) that is highly expressed in pediatric sarcomas, to repress critical tumor suppressors. We have developed a potent reversible LSD1 inhibitor, SP-2577 (Seclidemstat), that shows the ability to impair tissue culture cell viability in multiple ES cell lines and is currently in clinical trials. A more favorable therapeutic response to SP-2577 may be obtained by combining drugs that target multiple pathways and/or inhibit resistance mechanisms. Experimental Design: In this study, we applied an arrayed CRISPR screening (Synthego) to identify survival genes that can be candidates for molecularly targeted drugs. Gene candidates were validated using western blot, qPCR, soft agar assay. Ewing’s Sarcoma A673 ABL2i knockdown and scramble cell lines were generated, implanted into nude mice, and treated with SP-2577. Lastly, combinational drug treatments using FDA-approved inhibitors with SP-2577 were tested in 2D and 3D cultures. Results: CRISPR druggable library screen identified that downregulation of several genes resulted in increased SP-2577 cytotoxicity in multiple ES cell lines. Importantly, ABL2 knockdown was found to have a significant synergy in combination with SP-2577 treatment, both in vitro and in vivo. siRNA knockdown of ABL2 in combination with SP-2577 treatment revealed an increase in JNK signaling and Cleaved Caspases-3 activation with decreased Src phosphorylation. This data suggests that downregulation of ABL2 in combination with SP-2577 treatment promotes activation of apoptosis pathways in ES. In addition, combination treatment of SP-2577 with drugs targeting ABL2 pathway showed high synergy in vitro. Conclusion: This study highlights the potential for a combination treatment of SP-2577 with ABL2 inhibitors in ES patients. Further, our observations support the capability of arrayed CRISPR screening to identify new therapeutic strategies in ES. Citation Format: Samuel Sampson, Trason Thode, Kevin Drenner, Ryan Rodriguez Del Villar, Annika Ozols, Mohan Kaadige, Alexis Weston, Serina Ng, Tithi Ghosh Halder, Shelby Rheinschmidt, David Anderson, Kevin Holden, Raffaella Soldi, Sunil Sharma. Synergistic drug combination of LSD1 and ABL2 inhibitors for Ewing's sarcoma identified in an arrayed CRISPR screen approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1048.

Discovery of novel sulphonamide hybrids that inhibit LSD1 against bladder cancer cells

ABSTRACT Aim:A series of sulphonamide hybrids were designed, synthesised, and identified as potential lysine-specific demethylase 1 (LSD1) inhibitors. Materials and methods: Bladder cancer cell lines were cultured to evaluate the antiproliferative activity. Inhibitory evaluation of sulphonamide hybrids against LSD1 were performed. Conclusion: sulphonamide derivative L8 exhibited the antiproliferative activity against HTB5, HTB3, HT1376, and HTB1 cells with IC50 values of 1.87, 0.18, 0.09, and 0.93 μM, respectively. Compound L8 as a selective and reversible LSD1 inhibitor could inhibit LSD1 with the IC50 value of 60 nM. It effectively inhibited LSD1 by increasing the expression levels of H3K4me1, H3K4me2, and H3K9me2 in HT1376 cells. To the best of our knowledge, this was the first report which showed that sulphonamide–quinoline–dithiocarbamate hybrids potently inhibited LSD1 in bladder cancer cells. Our studies give the potential application of the sulphonamide-based scaffold for developing LSD1 inhibitors to treat bladder cancer.

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 > 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 ( > 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.

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 (>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]

Phase I Study of Lysine-Specific Demethylase 1 Inhibitor, CC-90011, in Patients with Advanced Solid Tumors and Relapsed/Refractory Non-Hodgkin Lymphoma

Lysine-specific demethylase 1 (LSD1) is implicated in multiple tumor types, and its expression in cancer stem cells is associated with chemoresistance. CC-90011 is a potent, selective, and reversible oral LSD1 inhibitor. We examined CC-90011 in advanced solid tumors and relapsed/refractory (R/R) non-Hodgkin lymphoma (NHL). CC-90011-ST-001 (NCT02875223; 2015-005243-13) is a phase I, multicenter, first-in-human dose-escalation study. Nine dose levels of CC-90011 (1.25-120 mg) given once per week were explored. Primary objectives were to determine safety, maximum tolerated dose (MTD), and/or recommended phase II dose (RP2D). Secondary objectives were to evaluate preliminary efficacy and pharmacokinetics. Fifty patients were enrolled, 49 with solid tumors (27 neuroendocrine tumors/carcinomas) and 1 with R/R NHL. Median age was 61 years (range, 22-75). Patients received a median of three (range, 1-9) prior anticancer regimens. The RP2D was 60 mg once per week; the nontolerated dose (NTD) and MTD were 120 mg once per week and 80 mg once per week, respectively. Grade 3/4 treatment-related toxicities were thrombocytopenia (20%; an on-target effect unassociated with clinically significant bleeding), neutropenia (8%; in the context of thrombocytopenia at the highest doses), and fatigue (2%). The patient with R/R NHL had a complete response, currently ongoing in cycle 34, and 8 patients with neuroendocrine tumors/carcinomas had stable disease ≥6 months, including bronchial neuroendocrine tumors, kidney tumor, and paraganglioma. CC-90011 is well tolerated, with the RP2D established as 60 mg once per week. The MTD and NTD were determined to be 80 mg once per week and 120 mg once per week, respectively. Further evaluation of CC-90011 is warranted.

Macrocyclic Peptides Uncover a Novel Binding Mode for Reversible Inhibitors of LSD1.

Lysine-specific demethylase 1 (LSD1) is an epigenetic enzyme which regulates the methylation of Lys4 of histone 3 (H3) and is overexpressed in certain cancers. We used structures of H3 substrate analogues bound to LSD1 to design macrocyclic peptide inhibitors of LSD1. A linear, Lys4 to Met-substituted, 11-mer (4) was identified as the shortest peptide distinctly interacting with LSD1. It was evolved into macrocycle 31, which was >40 fold more potent (Ki = 2.3 μM) than 4. Linear and macrocyclic peptides exhibited unexpected differences in structure–activity relationships for interactions with LSD1, indicating that they bind LSD1 differently. This was confirmed by the crystal structure of 31 in complex with LSD1-CoREST1, which revealed a novel binding mode at the outer rim of the LSD1 active site and without a direct interaction with FAD. NMR spectroscopy of 31 suggests that macrocyclization restricts its solution ensemble to conformations that include the one in the crystalline complex. Our results provide a solid basis for the design of optimized reversible LSD1 inhibitors.

Investigating the Binding Mode of Reversible LSD1 Inhibitors Derived from Stilbene Derivatives by 3D-QSAR, Molecular Docking, and Molecular Dynamics Simulation.

Overexpression of lysine specific demethylase 1 (LSD1) has been found in many cancers. New anticancer drugs targeting LSD1 have been designed. The research on irreversible LSD1 inhibitors has entered the clinical stage, while the research on reversible LSD1 inhibitors has progressed slowly so far. In this study, 41 stilbene derivatives were studied as reversible inhibitors by three-dimensional quantitative structure–activity relationship (3D-QSAR). Comparative molecular field analysis (CoMFA = 0.623, = 0.987, = 0.857) and comparative molecular similarity indices analysis (CoMSIA = 0.728, = 0.960, = 0.899) were used to establish the model, and the structure–activity relationship of the compounds was explained by the contour maps. The binding site was predicted by two different kinds of software, and the binding modes of the compounds were further explored. A series of key amino acids Val288, Ser289, Gly314, Thr624, Lys661 were found to play a key role in the activity of the compounds. Molecular dynamics (MD) simulations were carried out for compounds 04, 17, 21, and 35, which had different activities. The reasons for the activity differences were explained by the interaction between compounds and LSD1. The binding free energy was calculated by molecular mechanics generalized Born surface area (MM/GBSA). We hope that this research will provide valuable information for the design of new reversible LSD1 inhibitors in the future.

Small molecule inhibition of lysine-specific demethylase 1 (LSD1) and histone deacetylase (HDAC) alone and in combination in Ewing sarcoma cell lines.

Ewing Sarcoma (ES) is characterized by recurrent translocations between EWSR1 and members of the ETS family of transcription factors. The transcriptional activity of the fusion oncoprotein is dependent on interaction with the nucleosome remodeling and deactylase (NuRD) co-repressor complex. While inhibitors of both histone deacetylase (HDAC) and lysine-specific demethylase-1 (LSD1) subunits of the NuRD complex demonstrate single agent activity in preclinical models, combination strategies have not been investigated. We selected 7 clinically utilized chemotherapy agents, or active metabolites thereof, for experimentation: doxorubicin, cyclophosphamide, vincristine, etoposide and irinotecan as well as the HDAC inhibitor romidepsin and the reversible LSD1 inhibitor SP2509. All agents were tested at clinically achievable concentrations in 4 ES cell lines. All possible 2 drug combinations were then tested for potential synergy. Order of addition of second-line conventional combination therapy agents was tested with the addition of SP2509. In two drug experiments, synergy was observed with several combinations, including when SP2509 was paired with topoisomerase inhibitors or romidepsin. Addition of SP2509 after treatment with second-line combination therapy agents enhanced treatment effect. Our findings suggest promising combination treatment strategies that utilize epigenetic agents in ES.

Abstract 3833: Antitumor activity of novel reversible LSD1 inhibitor in preclinical models of AML and SCLC

Abstract Introduction: Global changes in the epigenetic landscape are known as the hallmark in cancer and the histone demethylase Lysine-specific demethylase 1 (LSD1) is one of the novel targets for the therapy of various malignant diseases. Also LSD1 overexpression is associated with poor prognosis in various cancers. LSD1-mediated epigenetic modification is known to play a key role in the regulation of gene expression by removing the methyl groups from methylated lysine 4 and lysine 9 of histone H3. Alterations in histone methylation lead to aberrant silencing of expression of multiple genes involved in tumor survival and in cell cycle. Moreover, epigenetic dysregulation plays a critical role in pathogenesis of various types of cancers such as acute myeloid leukemia (AML) or small cell lung cancer (SCLC). In this study, we have characterized a novel LSD1 inhibitor and assessed its potential as a novel therapy for AML and SCLC patients. Materials and Methods: Novel LSD1 inhibitor, HM97211 and its derivatives were designed and synthesized as the active biologic inhibitory compound against the AML and SCLC cells. Biochemical enzyme assay was performed to identify enzyme selectivity of HM97211 derivatives. Standard proliferation assay, immunoblotting, and apoptosis analysis were carried out to validate the potency of HM97211 derivatives in AML and SCLC cell lines. In vivo anti-tumor activity was evaluated in AML and SCLC cell xenograft mice models. Tumor sizes were measured and tumor samples were analyzed to define the mechanisms of action. Results: HM97211 derivatives showed higher selectivity toward LSD1 compared to other FAD-utilizing enzyme as well as other epigenetic enzymes. HM97211 derivatives alter expression of various genes and induce differentiation and apoptosis, resulting in growth suppression in a panel of AML and SCLC cells. Moreover, HM97211 derivatives significantly induced programmed cell death in AML and SCLC cells. Oral administration of HM97211 derivatives inhibited tumor growth of human AML and SCLC xenograft models as a single agent at doses exhibiting significant. Efficacy of HM97211 derivatives was further evaluated in combination treatment with standard chemotherapies in human AML and SCLC xenograft model. Conclusion: Collectively, results of this study suggested that novel reversible LSD1 inhibitor, HM97211 derivatives, can be a strong therapeutic agent for AML and SCLC patients. Hanmi presents a new insight to epigenetics application in anticancer therapy. Citation Format: InHwan Bae, WonJeoun Kim, JiSook Kim, JiYoung Song, JungSoo Nam, Moonsub Lee, Jooyun Byun, Hyeongki Kim, TaeHun Song, Seokhyun Hong, KyuHang Lee, YoungGil Ahn, YoungHoon Kim, Kwee Hyun Suh. Antitumor activity of novel reversible LSD1 inhibitor in preclinical models of AML and SCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3833.

Identification of selective and reversible LSD1 inhibitors with anti-metastasis activity by high-throughput docking

The overexpression of lysine specific demethylase 1 (LSD1) has been reported in various human tumors. There is increasing interest in targeting LSD1 with small molecules for cancer treatment. A released structure of an LSD1 kinase domain in complex with FAD was used to set up a low-cost high-throughput docking protocol for quick identification of LSD1 inhibitors. The most promising hit L05 was confirmed to be a potent, selective and reversible LSD1 inhibitor and displayed marked inhibition of colorectal cells migration without significant cytotoxicity.

Preclinical studies reveal that LSD1 inhibition results in tumor growth arrest in lung adenocarcinoma independently of driver mutations.

Lung adenocarcinoma (LUAD) is the most prevalent subtype of non‐small cell lung cancer. Despite the development of novel targeted and immune therapies, the 5‐year survival rate is still only 21%, indicating the need for more efficient treatment regimens. Lysine‐specific demethylase 1 (LSD1) is an epigenetic eraser that modifies histone 3 methylation status, and is highly overexpressed in LUAD. Using representative human cell culture systems and two autochthonous transgenic mouse models, we investigated inhibition of LSD1 as a novel therapeutic option for treating LUAD. The reversible LSD1 inhibitor HCI‐2509 significantly reduced cell growth with an IC 50 of 0.3–5 μm in vitro, which was linked to an enhancement of histone 3 lysine methylation. Most importantly, growth arrest, as well as inhibition of the invasion capacities, was independent of the underlying driver mutations. Subsequent expression profiling revealed that the cell cycle and replication machinery were prominently affected after LSD1 inhibition. In addition, our data provide evidence that LSD1 blockade significantly interferes with EGFR downstream signaling. Finally, our in vitro results were confirmed by preclinical therapeutic approaches, including the use of two autochthonous transgenic LUAD mouse models driven by either EGFR or KRAS mutations. Importantly, LSD1 inhibition resulted in significantly lower tumor formation and a strong reduction in tumor progression, which were independent of the underlying mutational background of the mouse models. Hence, our findings provide substantial evidence indicating that tumor growth of LUAD can be markedly decreased by HCI‐2509 treatment, suggesting its use as a single agent maintenance therapy or combined therapeutical application in novel concerted drug approaches.

Optimization of 5-arylidene barbiturates as potent, selective, reversible LSD1 inhibitors for the treatment of acute promyelocytic leukemia

Histone lysine specific demethylase 1 (LSD1) is overexpressed in diverse hematologic disorders and recognized as a promising target for blood medicines. In this study, molecular docking-based virtual screening united with bioevaluation was utilized to identify novel skeleton of 5-arylidene barbiturate as small-molecule inhibitors of LSD1. Among the synthesized derivatives, 12a exhibited reversible and potent inhibition (IC50 = 0.41 μM) and high selectivity over the MAO-A and MAO-B. Notably, 12a strongly induced differentiation effect on acute promyelocytic leukemia NB4 cell line and distinctly escalated the methylation level on histone 3 lysine 4 (H3K4). Our findings indicate that 5-arylidene barbiturate may represent a new skeleton of LSD1 inhibitors and 12a deserve as a promising agent for the further research.

Investigating the binding mechanism of (4-Cyanophenyl)glycine derivatives as reversible LSD1 by 3D-QSAR, molecular docking and molecular dynamics simulations

Lysine specific demethylase 1 (LSD1) have been regard as an important drug target on the therapy of cancer and leukemia. However, the development of effective reversible LSD1 inhibitors was facing many challenges. In this work, we carried out a molecular modeling study on (4-Cyanophenyl)glycine derivatives as reversible LSD1 inhibitors using 3D-QSAR, molecular docking and molecular dynamics simulations. Molecular docking study revealed the possible binding mechanism of these inhibitors with LSD1. We used comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) to generate 3D-QSAR models. The results showed that our CoMFA model had q2 of 0.785, r2 = 0.994 and r2pred of 0.92, while the best CoMSIA model had q2 of 0.746, r2 = 0.985 and r2pred of 0.86. Molecular dynamics simulations validated the rationality of docking results and predicted the detailed interactions between the ligands and LSD1. An important hydrogen bond network was discovered though MD simulation. Some key residues (FAD, Asp555, Gln358, Tyr761, Lys661 and Trp695) were pointed out after the binding free energy calculation using MM-PBSA method. We hope these result could provide useful information for our further design of potent and selective LSD1 inhibitors.

Abstract 4871: Antitumor activity of novel reversible LSD1 inhibitor, HM97211 in preclinical models of SCLC

Abstract Introduction: Global changes in the epigenetic landscape are known as the hallmark in cancer and the histone demethylase Lysine-specific demethylase 1 (LSD1) is one of the novel target for the therapy of various malignant diseases and LSD1 overexpression is associated with poor prognosis in various cancers. LSD1-mediated epigenetic modification is known to play a key role in the regulation of gene expression by removing the methyl groups from methylated lysine 4 and lysine 9 of histone H3. Alterations in histone methylation lead to aberrant silencing of expression of multiple genes involved in tumor survival and in cell cycle. Moreover, epigenetic dysregulation plays a critical role in pathogenesis of various types of cancers such as acute myeloid leukemia (AML) or small cell lung cancer (SCLC). Herein, Hanmi introduces, a safe drug profile of the novel LSD1 inhibitor, H97211on hematopoiesis differentiation by reversible binding on LSD1. Materials and Methods: Novel reversible LSD1 inhibitor, HM97211 was designed and synthesized as the active biologic inhibitory compound against the SCLC cells. The binding mode on reversibility for HM97211 and biochemical activity on LSD1 were determined by LSD1 Fluorescent assay kit (BPS Bioscience; Venkataswamy Sorna et al, 2013). In vitro proliferation assay was performed by using the CellTiter-Glo (Promega) In vivo therapeutic efficacy of HM97211 was evaluated in NCI-H1417 xenograft mice models. 5 x 106 NCI-H1417 cells/mouse were implanted subcutaneously and mice were treated by daily oral administration of (30mg/kg) HM97211. Tumor sizes were measured and tumor samples were analyzed of the mechanisms of action. Results: HM97211 enhanced not only global methylation of H3K4 but also apoptotic signaling in NCI-H1417 cell line. HM97211 altered expression of neuroendocrine factor ASCL1, inducing programmed cell death in SCLC cells. In NCI-H1417 xenograft models, daily oral administration of HM97211 induced tumor regression without remarkable toxicity. Analyses of tumor samples revealed the important role of potent small molecule LSD1 inhibitor in neuroendocrine-associated transcription and cell proliferation of SCLC. Conclusion: Collectively, results of this study suggested that novel reversible LSD1 inhibitor, HM97211, can be a strong therapeutic agent for SCLC patients. Hanmi presents a new insight to epigenetics application in anticancer therapy. Citation Format: InHwan Bae, JiSook Kim, ChangHee Park, Hyeongki Kim, Namgoong Gwangmog, Joo-Yun Byun, Seokhyun Hong, Teahun Song, JaeHo Lee, Kyuhang Lee, Myoungsil Ko, Cheolkyung Kim, YoungMi Lee, Ho Jeong Lee, YoungHoon Kim, YoungGil Ahn, KweeHyun Suh, Sun-Jin Kim. Antitumor activity of novel reversible LSD1 inhibitor, HM97211 in preclinical models of SCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4871.

Abstract 4873: Therapeutic targeting of LSD1 in Ewing sarcoma with SP-2509

Abstract Objective: Multimodal regimes remain the cornerstone treatment for Ewing sarcoma (ES), the second most common solid bone malignancy diagnosed in pediatric and young adolescent populations. We have reached a therapeutic ceiling with conventional cytotoxic agents, highlighting the need to adopt novel approaches specifically targeting the drivers of ES oncogenesis. As LSD1 (Lysine Specific Demethylase 1) is highly expressed in ES tumors, with elevated expression levels associated with worse overall survival, this study examined biomarkers of sensitivity/mechanisms of cytotoxicity to targeted LSD1 inhibition using SP-2509 (reversible LSD1 inhibitor). Methods: The antiproliferative effects of SP-2509 were determined through Cell Titre Glo assays following 72hrs of treatment in a panel of 17 ES cell lines with varying STAG2/TP53 mutational status and basal LSD1 expression levels. RNA-seq analysis of six ES cell lines -/+ SP-2509 treatment (2μM) was also conducted. Results: Innate resistance to SP-2509 was not observed in our cell line cohort (72hr IC50:81nM-1593nM). In contrast, resistance to the clinical next-generation LSD1 irreversible inhibitor GSK-LSD1 was observed across multiple cell lines (144hr IC50>300μM). Although TP53/STAG2 mutational status and basal LSD1 mRNA/protein levels did not correlate with SP-2509 response, induction of LSD2 (homologue of LSD1) following SP-2509 treatment was strongly associated with SP-2509 hypersensitivity (R2=0.562). shRNA mediated knockdown of LSD2 significantly reduced the cytotoxic effects of SP-2509 (4.3 fold IC50 increase) only in hypersensitive cell lines. Mechanistically, RNA-seq analysis revealed that SP-2509 imparts apoptosis through engagement of the endoplasmic reticulum stress pathway, with hypersensitive cell lines sharing similar transcriptomic profiles. In addition, ETS1/HIST1H2BM were specifically induced/repressed, respectively, following SP-2509 treatment only in our hypersensitive cell lines. Finally, we demonstrate that the transcriptional profile driven by SP-2509 strongly mirrors LSD1 genetic depletion. Conclusion: Our findings provide key insights into the mechanisms of SP-2509 cytotoxicity as well as biomarkers that can be used to predict LSD1 inhibitor sensitivity in ES. Citation Format: Kathleen Pishas, Megann Boone, Sunil Sharma, Stephen Lessnick. Therapeutic targeting of LSD1 in Ewing sarcoma with SP-2509 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4873.

Abstract LB-240: LSD1 activates a lethal prostate cancer gene network independently of its demethylase function

Abstract Background: Medical castration that interferes with androgen receptor (AR) function is the principal treatment for advanced prostate cancer. However, clinical progression is universal, and tumors with AR-independent resistance mechanisms appear to be increasing in frequency. Consequently, there is an urgent need to develop new treatments targeting molecular pathways enriched in lethal prostate cancer. Lysine specific demethylase 1 (LSD1) is a histone demethylase and important regulator of gene expression. Here we describe a distinct role of LSD1 as a driver of proliferation and survival of castration-resistant prostate cancer (CRPC) cells independently of its demethylase function and of the AR. Methods: We used loss of function studies to determine the importance of LSD1 for survival of prostate cancer cells. To identify transcriptional networks that contribute to cell survival, we suppressed LSD1 with RNAi and performed gene expression profiling. To determine the importance of LSD1's demethylase function in regulation of target genes, we measured LSD1's canonical histone substrates (H3K4me2 and H3K9me2) using genome-wide chromatin immunoprecipitation-sequencing and measured the effect of ectopically expressed wild type or catalytically deficient LSD1 on cell survival and target gene expression. We used mass spectrometry (MS) to identify important LSD1 protein complex members. Finally, we used multiple biochemical and cellular assays and an in vivo study to confirm the mechanism of action of a small molecule inhibitor that targets critical, non-canonical functions of LSD1 in CRPC cells. Results: Cell viability assays demonstrated that LSD1 is important for proliferation and survival of CRPC cells independently of the AR. Gene expression profiles demonstrated that LSD1 activates androgen-independent genes that are enriched in lethal human tumors. Importantly, our global epigenomic studies and biochemical experiments demonstrated that LSD1's demethylase function was dispensable for these effects, while our MS studies identified cooperating LSD1 binding proteins. Finally, we identified a reversible small molecule inhibitor of LSD1 that blocked critical, demethylase-independent functions in vitro and in vivo. Conclusions: Our results demonstrate that LSD1 promotes survival of prostate cancer cells, including those that are castration-resistant, independently of its demethylase function and of the AR. Importantly, this effect is explained in part by activation of a lethal prostate cancer gene network in collaboration with specific binding proteins. Finally, that a reversible small molecule LSD1 inhibitor blocks important demethylase-independent functions and suppresses CRPC cell viability demonstrates the potential of LSD1 inhibition in this disease. Citation Format: Archana Sehrawat, Dae-Hwan Kim, Lina Gao, Yuliang Wang, Armand Bankhead, Shannon McWeeney, Carly J. King, Jacob Schwartzman, Junior Tayou, Joshua Urrutia, William H. Bisson, Daniel J. Coleman, Sunil K. Joshi, David A. Sampson, Sheila Weinmann, Bhaskar VS Kallakury, Deborah L. Berry, Reina Haque, Stephen K. Van Den Eeden, Sunil Sharma, Jared Bearss, Tomasz M. Beer, George V. Thomas, Laura M. Heiser, Joshi J. Alumkal. LSD1 activates a lethal prostate cancer gene network independently of its demethylase function [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-240.

Crystal Structure of LSD1 in Complex with 4-[5-(Piperidin-4-ylmethoxy)-2-(p-tolyl)pyridin-3-yl]benzonitrile.

Because lysine-specific demethylase 1 (LSD1) regulates the maintenance of cancer stem cell properties, small-molecule inhibitors of LSD1 are expected to be useful for the treatment of several cancers. Reversible inhibitors of LSD1 with submicromolar inhibitory potency have recently been reported, but their exact binding modes are poorly understood. In this study, we synthesized a recently reported reversible inhibitor, 4-[5-(piperidin-4-ylmethoxy)-2-(p-tolyl)pyridin-3-yl]benzonitrile, which bears a 4-piperidinylmethoxy group, a 4-methylphenyl group, and a 4-cyanophenyl group on a pyridine ring, and determined the crystal structure of LSD1 in complex with this inhibitor at 2.96 Å. We observed strong electron density for the compound, showing that its cyano group forms a hydrogen bond with Lys661, which is a critical residue in the lysine demethylation reaction located deep in the catalytic center of LSD1. The piperidine ring interacts with the side chains of Asp555 and Asn540 in two conformations, and the 4-methylphenyl group is bound in a hydrophobic pocket in the catalytic center. Our elucidation of the binding mode of this compound can be expected to facilitate the rational design of more-potent reversible LSD1 inhibitors.