Potent LSD1 Inhibitors Research Articles

Design, Synthesis, and Evaluation of the Selective and Orally Active LSD1 Inhibitor with the Potential of Treating Heart Failure.

LSD1 has become an appealing target for the development of new pharmacologic agents to treat cardiovascular diseases, including heart failure. Herein, we reported the design, synthesis, and structure-activity relationship of a series of TCP-based derivatives targeting LSD1. Docking studies were employed to successfully elucidate the SAR. Particularly, compound 7d, characterized by low toxicity, demonstrated a high affinity for LSD1 at molecular and cellular levels. It also displayed favorable pharmacokinetic properties for oral dosing (e.g., F = 77.61%), effectively alleviating Ang II-induced NRCFs activation in vitro and reducing pathological myocardial remodeling in TAC-induced cardiac remodeling and heart failure in vivo. Additionally, mechanism studies revealed that suppression of myocardial dysfunction by compound 7d is related to LSD1 inhibition-induced TGFβ signaling pathway repressing. In summary, the current report presents compound 7d as a potent LSD1 inhibitor with the potential for further development as a therapeutic agent for pressure overload-related heart failure.

Abstract B025: ASCL1 and SOX2 expression levels predict sensitivity to LSD1 inhibition with iadademstat in small cell lung cancer

Abstract Lysine Specific Demethylase 1 (LSD1, also known as KDM1A) is a member of the FAD-containing family of lysine demethylases and is generally involved in repressing transcription by specifically removing methyl groups from mono- and di-methylated lysine 4 of histone 3 - epigenetic marks commonly associated with actively transcribed genes. LSD1 over-expression has been associated with disease progression and worse prognosis in several human cancers, and its inhibition has been shown to reduce cancer cell growth, migration and invasion. LSD1 has therefore been recognized as a target of interest for the development of new drugs to treat cancer. Iadademstat (aka ORY-1001), a highly potent and selective LSD1 inhibitor (LSD1i) in clinical development for malignant indications, and other LSD1is, have been reported to be effective for the treatment of small cell lung cancer (SCLC) in in vitro and in vivo models. However, the published data indicate that while certain SCLC are highly sensitive to LSD1 inhibition this sensitivity is not a universal feature of SCLC cells raising the need to develop methods for identifying sensitive SCLC tumors. Herein, we report the identification of the predictive biomarkers ASCL1 and SOX2 for the enrichment of the SCLC population more likely to respond to iadademstat and other LSD1is. Response to LSD1is was evaluated in a broad panel of SCLC cell lines (from viability assay data obtained internally at Oryzon Genomics with iadademstat and from data published elsewhere using other LSD1is). Additionally, analysis of gene expression was performed in this cell line panel using internal and publicly available datasets (Affymetrix, CCLE). This analysis revealed that the transcription factors ASCL1 and SOX2 are more likely to be highly expressed in SCLC cell lines that respond to LSD1 inhibition. Moreover, the mRNA levels of these biomarkers were highly correlated with their protein levels in both SCLC cell lines and in patient-derived xenografts. In particular, immunofluorescence staining of these patient-derived SCLC samples can discriminate different degrees of ASCL1 and SOX2 protein expression levels (none, low, medium, high), which correlate with their mRNA levels determined by RNAseq in the same samples. Moreover, analysis of ASCL1 and SOX2 protein levels analyzed internally by immunofluorescence in 40 SCLC biopsies showed that 58% samples concomitantly expressed medium to high levels of both biomarkers. Taken together, these data suggest that medium to high expression of both ASCL1 and SOX2 define a SCLC population more likely to respond to LSD1 inhibition, and that this population could account for nearly 60% of SCLC cases. Therefore, ASCL1 and SOX2 expression could be used as predictive biomarkers for personalized medicine with iadademstat in SCLC patients and clinical research to validate this hypothesis is warranted. Citation Format: Natalia Sacilotto, Serena Lunardi, Filippo Ciceri, Cristina Mascaro, Tamara Maes, Ana Limon, Douglas V. Faller. ASCL1 and SOX2 expression levels predict sensitivity to LSD1 inhibition with iadademstat in small cell lung cancer. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr B025.

Design and Synthesis of Benzene Homologues Tethered with 1,2,4-Triazole and 1,3,4-Thiadiazole Motifs Revealing Dual MCF-7/HepG2 Cytotoxic Activity with Prominent Selectivity via Histone Demethylase LSD1 Inhibitory Effect.

In this study, an efficient multistep synthesis of novel aromatic tricyclic hybrids incorporating different biological active moieties, such as 1,3,4-thiadiazole and 1,2,4-triazole, was reported. These target scaffolds are characterized by having terminal lipophilic or hydrophilic parts, and their structures are confirmed by different spectroscopic methods. Further, the cytotoxic activities of the newly synthesized compounds were evaluated using in vitro MTT cytotoxicity screening assay against three different cell lines, including HepG-2, MCF-7, and HCT-116, compared with the reference drug Taxol. The results showed variable performance against cancer cell lines, exhibiting MCF-7 and HepG-2 selectivities by active analogs. Among these derivatives, 1,2,4-triazoles 11 and 13 and 1,3,4-thiadiazole 18 were found to be the most potent compounds against MCF-7 and HepG-2 cancer cells. Moreover, structure–activity relationship (SAR) studies led to the identification of some potent LSD1 inhibitors. The tested compounds showed good LSD1 inhibitory activities, with an IC50 range of 0.04–1.5 μM. Compounds 27, 23, and 22 were found to be the most active analogs with IC50 values of 0.046, 0.065, and 0.074 μM, respectively. In addition, they exhibited prominent selectivity against a MAO target with apparent cancer cell apoptosis, resulting in DNA fragmentation. This research provides some new aromatic-centered 1,2,4-triazole-3-thione and 1,3,4-thiadiazole analogs as highly effective anticancer agents with good LSD1 target selectivity.

P586: IADADEMSTAT COMBINATION WITH AZACITIDINE SHOWS ENCOURAGING SAFETY AND EFFICACY DATA IN ELDERLY AND UNFIT AML PATIENTS

Background: Acute Myeloid Leukemia (AML) is an hematological malignancy with highest incidence and lower survival rates in elderly. Previously reported ORR with hypomethylating agents such as azacitidine alone is less than 30%. Recently, combinations of hypomethylating agents with venetoclax have shown improved response ratios. However, refractoriness or relapse is still a challenge for most patients (pts), particularly in the elderly and the high-risk subpopulations. It has been shown that LSD1 is involved in malignant transformation in AML. Iadademstat (iada) is a potent and selective LSD1 inhibitor that has been administered to more than 100 oncology pts in Ph1 and Ph2 trials, showing manageable toxicity and signs of preliminary activity. Aims: ALICE (EudraCT 2018-000482-36) is a Phase IIa study to assess the safety, tolerability and the recommended Phase II dose (RP2D) of iada (PO 5d ON, 2d OFF every week in 28d cycles) in combination with azacitidine (sc., d1-5 and 8-9 every cycle) for the treatment of adult patients diagnosed with AML, as per WHO 2016 classification, who have not received prior treatment and are ineligible for intensive chemotherapy. We present an update of the ongoing study six months after completion of recruitment. Methods: Two doses of iada are studied in the trial: 60 and 90µg/m2/d. Besides the safety and tolerability primary endpoints, secondary endpoints investigate anti-leukemic activity including ORR according to ELN recommendations, time to response (TTR) and duration of responses (DoR). Additional assessments include residual detectable disease status, overall survival and PK/PD determinations. Results: At EHA 2021, ALICE data from 27 pts were reported. In October 2021, recruitment of the targeted 36 pts was completed. Patient baseline characteristics are shown in Table 1. Main safety events by the end of 2021 included 348 adverse reactions (AR). The most frequently reported AR was platelet reduction, observed in 44% of pts, however, Grade ≥3 thrombocytopenia was already present at baseline in 61% of pts (Table 1). Only three Grade 3-4 nonhematological treatment-related ARs were observed in two pts (asthenia, dysgeusia and weight decrease). Among the 71 serious AR reported, only 2 were considered as potentially related to iada, corresponding to a differentiation syndrome and a fatal intracranial hemorrhage, previously presented at EHA 2021. By the time this abstract is written, among the pts intended to treat (34), 27 have had at least 1 bone marrow evaluation after cycle 1 and are therefore evaluable for efficacy. 78% of them (21 of 27) achieved an objective response; of which, 62% were CR/CRi. Moreover, 80% of the CRi pts also achieved partial hematology recovery (CRh). Among CR/CRi pts, 5 out of the 8 already assessed by MRD presented no residual detectable disease by flow cytometry. DoR is encouraging with 77% of the CR/CRi lasting more than 6 months, with a mean DoR increasing as the study progresses, with the longest CR to date above 1,100 days. Iada at 90 µg/m2/d is the confirmed RP2D for the combination, showing deep responses with manageable toxicity. Image:Summary/Conclusion: ALICE data confirms that the combination of iada with azacitidine gives robust, fast and durable responses in unfit, previously untreated, AML patients. Considering iada’s efficacy, pharmacologic properties, its manageable toxicity and low anticipated drug-drug interactions, iada combinations may offer additional therapeutic options for AML patients in first line or in the R/R setting.

The novel LSD1 inhibitor ZY0511 suppresses diffuse large B-cell lymphoma proliferation by inducing apoptosis and autophagy

Lysine-specific demethylase 1 (LSD1, also known as KDM1A) is an attractive agent for treatment of cancer. However, the anti-tumor effect of LSD1 inhibitors against diffuse large B-cell lymphoma (DLBCL) and the underlying mechanism are still unclear. Here, we report that KDM1A is overexpressed in human DLBCL tissues and negatively related to overall survival rate of DLBCL patients. ZY0511, a novel and potent LSD1 inhibitor developed by our group, inhibited the proliferation of human DLBCL cells. ZY0511 interacted with LSD1, induced methylation level of histone 3 lysine 4 and histone 3 lysine 9 in DLBCL cells. Mechanistically, transcriptome sequencing results indicated that ZY0511 induced the genes enrichment significantly related to cell cycle, autophagy, and apoptosis signaling pathways. Further study confirmed that ZY0511 blocked cell cycle at G0/G1 phase and expression of CDK4 and cyclin D1. ZY0511 decreased mitochondrial membrane potential and induced apoptosis, which can be reverted by a pan-caspase inhibitor, Z-VAD-FMK. Moreover, ZY0511 treatment significantly increased autophagy-associated marker proteins and autophagosomes formation in DLBCL cells. In vivo xenograft experiments confirmed that intraperitoneal administration of ZY0511 significantly suppressed SU-DHL-6 xenograft tumor growth in vivo. In conclusion, our findings identify that ZY0511 inhibits DLBCL growth both in vitro and in vivo via the induction of apoptosis and autophagy, and LSD1 inhibitor might be a promising strategy for treating DLBCL.

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.

Discovery of higenamine as a potent, selective and cellular active natural LSD1 inhibitor for MLL-rearranged leukemia therapy

Natural products are a rich source of lead compounds and have shown promise for epigenetic drug discovery. In this work, we discovered higenamine from our natural product library as a potent, selective and cellular active natural LSD1 inhibitor. Higenamine shows acceptable potency against LSD1 and high selectivity towards LSD1 over MAOA/B. Higenamine significantly increases expression of LSD1 substrates H3K4me1 and H3K4me2 in MLL-rearranged leukemia cells MV4-11 and MOLM-13, but nearly had no effect on LSD1 and H3K4Me3. Meanwhile, higenamine dose-dependently suppresses the levels of HOXA9 and MEIS1 that are overexpressed in leukemia cell lines. Notably, higenamine induces cell differentiation of MV4-11 and MOLM-13 cells accompanying by increased expression of CD11b, CD14 and CD86. Higenamine promotes cell apoptosis, inhibits colony formation, but does not inhibit proliferation of leukemia cells significantly. In addition, the expression levels of p53 are dramatically changed by higenamine in an LSD1-dependent manner in MV4-11 cells. Taken together, higenamine could be employed as a starting point for the development of more selective and potent LSD1 inhibitors. Our work firstly reveals the non-classical epigenetic regulation mechanism of higenamine in cancers, and also demonstrates the efficacy of higenamine for MLL-rearranged leukemia therapy.

Identification of fenoldopam as a novel LSD1 inhibitor to abrogate the proliferation of renal cell carcinoma using drug repurposing strategy

Although targeted therapy for renal cell carcinoma (RCC) has achieved good therapeutic effects in clinic, a considerable number of patients develop drug resistance over time. So, there is still an urgent need to develop new drugs for RCC treatment. As LSD1 is considered as a promising drug target in diverse cancers, including RCC, we tried to find new LSD1 inhibitor using drug repurposing strategy from a compound library, and fenoldopam, an FDA-approved drug, was identified as a potent LSD1 inhibitor with IC50 = 0.8974 μM in a reversible manner. Molecular docking predicted that fenoldopam occupied the FAD cavity of LSD1, forming hydrogen bonds with surrounding residues. Moreover, fenoldopam inactivated LSD1 and performed antiproliferative activity against ACHN cells and promoted cells apoptosis in vitro. Taken together, fenoldopam was identified as a novel LSD1 inhibitor firstly, and may serve as a new skeleton for RCC therapy.

4-Hydroxy-3-methylbenzofuran-2-carbohydrazones as novel LSD1 inhibitors

Histone lysine specific demethylase 1 (LSD1 or KDM1A) is a potential therapeutic target in oncology due to its overexpression in various human tumors. We report herein a new class of benzofuran acylhydrazones as potent LSD1 inhibitors. Among the 31 compounds prepared, 14 compounds exhibited excellent LSD1 inhibitory activity with IC50 values ranging from 7.2 to 68.8 nM. In cellular assays, several compounds inhibited the proliferations of various cancer cell lines, including PC-3, MCG-803, U87 MG, PANC-1, HT-29 and MCF-7. This opens up the opportunity for further optimization and investigation of this class compounds for potential cancer treatment.

PF277 ALICE: AN AML STUDY WITH LSD1 INHIBITION IN COMBINATION WITH AZACITIDINE IN THE ELDERLY

Background:Acute Myeloid Leukaemia (AML) is primarily a disease of older people and its incidence is rising. Survival rates decrease with age as does the achievement of complete remission (CR) with current chemotherapy, for which there is no consensus for optimal re‐therapy. Azacitidine is approved for AML with 20–30% blasts. Lysine‐specific demethylase (LSD1) has been shown to be a partner in some gene transformations in AML and helps sustain the oncogenic process. Iadademstat (ORY‐1001) is a highly potent and selective LSD1 inhibitor that has been shown to be effective in preclinical models (both alone or in combination with other compounds, including Azacitidine); and remarkably safe in a phase I study, where it also demonstrated preliminary anti‐leukaemic activity as monotherapy. Iadademstat in combination with azacitidine may thus offer a novel treatment option for a patient group with limited current options.Aims:To assess safety, tolerability and dose finding of iadademstat in combination with azacitidine, and to measure the clinical activity such as time to response (TTR), duration of response (DOR) and objective response (OR) of this combination in older patients who are not eligible to be treated with intensive chemotherapy. Other assessments include haematological improvement and overall survival, along with PK/PD measures (including a set of 6 blood biomarkers).Methods:This is a two‐stage phase II multicentre open‐label study of safety, tolerability, dose‐finding and efficacy of iadademstat in combination with azacitidine in elderly patients. The dose finding stage will dose 12–18 patients with a starting dose of iadademstat of 90 μg/m2/d. Depending on the DLTs observed, iadademstat may be escalated or de‐escalated. Once the Recommended Phase II Dose (RP2D) is identified, an expansion cohort of 18 patients will be enrolled and treated with iadademstat combined with azacitidine. In all cases patients to be recruited correspond to subjects ≥ 60 years of age with AML according to World Health Organization (WHO) classification, who are considered by the investigator to be ineligible for intensive chemotherapy regimen at that time or have refused standard chemotherapy, and which may not have received prior treatment for AML other than Hydroxyurea.Results:The patient assessments of all the enrolled study subjects will be presented as per May 2019 cut‐off, with particular emphasis on available preliminary efficacy outcomes. Current recruitment rate suggests that the available data will include the safety data after the first‐cycle of treatment of the first 8–10 participants. Moreover, preliminary efficacy data of all subjects in any of the cycles will be also presented.Summary/Conclusion:This first combination Phase IIa study with iadademstat aims to further extend the safety and efficacy of iadademstat in the oncology field; in this case, in older unfit leukemic patients as a first line treatment, providing initial support for the effect of its holistic unique MoA, which has been recently related to the potentiation of several other approaches, including immune‐oncology therapies in solid tumors.

Genome-Wide CRISPR-Cas9 Screen Identifies Sensitizers to LSD1 Inhibition in MLL-Translocated Human AML Cells

Lysine-specific demethylase (LSD1, also known as KDM1A) is an epigenetic regulator that has recently emerged as a potential therapeutic target in acute myeloid leukemia (AML). It is a flavin dependent monoamine oxidase which can demethylate monomethyl or dimethyl lysine 4 of histone H3. Pharmacological inhibition of LSD1 induces differentiation of blast cells in MLL-translocated AML and has shown significant promise in pre-clinical studies. With LSD1 inhibitors advancing through early-phase clinical trials, there is a strong pre-clinical rationale for the identification of genes whose protein products collaborate with LSD1 to retard differentiation in cancer and which could potentially be targeted in combination therapies for enhanced therapeutic benefit.To identify potential drug-gene synthetic lethal interactions, we performed a genome wide loss-of-function CRISPR-Cas9 screen in human THP1 AML cells treated with a potent and selective tranylcypromine-derivative LSD1 inhibitor trans-N-((2-methoxypyridin-3-yl)methyl)-2-phenylcyclopropan-1-amine (OG86). THP1 cells exhibit a t(9;11) MLL gene rearrangement and display similar phenotypic and functional responses to those observed in primary MLL-translocated AML cells following LSD1 inhibition. The cells were transduced with lentiviral human CRISPR knockout (hGeCKOv2) library containing 122,411 sgRNAs targeting 19,050 protein coding genes and 1864 miRNA precursors in the human genome. The transduced cells were divided into two groups, treated with either DMSO or 250nM OG86 and maintained in culture for ~10 population doublings. To investigate sgRNA representation in the cell population harvested at different stages of the screen, sgRNA cassettes were PCR amplified from genomic DNA and deep sequenced; negatively selected genes were identified with the MAGeCK computational algorithm. After successful initial quality assessment of the screen, we next searched for genes selectively depleted in OG86-treated versus DMSO-treated THP1 cells in samples collected on Day 15 and Day 18. At a false discovery rate of 7.5% there were 10 expressed genes whose sgRNA representation was depleted at both time points. In particular, these included two genes coding for components of the MTOR signaling pathway: MTOR associated protein, LST8 homolog (MLST8) and Ras-related GTP-binding protein A (RRAGA). In the Day 18 comparison, an additional MTOR pathway gene LAMTOR2 scored among the ten most depleted. MLST8 is a core component of TORC1/TORC2 complex and RRAGA is involved in the activation of TORC1 by amino acids. Recruitment of both RAG proteins and TORC1 to lysosomal membranes in response to amino acids, and the consequent activation of TORC1 signalling, requires the trimeric Ragulator complex, of which LAMTOR2 is a member.Based on our screen, we hypothesized that THP1 AML cells exposed to pharmacologic inhibition of LSD1 exhibit increased sensitivity to concomitant inhibition of the amino acid sensing component of the TORC1 pathway.Using genetic knockdown and pharmacological inhibition strategies, we then validated our screen hits in combination with LSD1 inhibition in targeting human AML cells. RNAi based knockdown of RRAGA, LAMTOR2 and MLST8 in combination with LSD1 inhibition were found to promote myeloid differentiation and reduce cell proliferation in THP1 cells. Interestingly the mTORC1 pathway inhibitor everolimus (RAD001) showed at least additive effect in combination with OG86 to decrease THP1 cell proliferation and promote immunophenotypic differentiation. Comparison of transcription changes in combined versus single treatment conditions by RNA-seq analysis further confirmed a more extensive and wide-ranging upregulation of a myeloid differentiation program upon concomitant inhibition of LSD1 and mTORC1 pathway. In vitro studies performed in primary patient AML cells gave similar results. Finally, in vivo studies using AML patient-derived xenograft mouse model confirmed that combination treatment promotes a strong myeloid differentiation program. In conclusion, we report here that inhibition of mTORC1 sensitizes human MLL-translocated AML cells to LSD1 inhibitor-mediated differentiation therefore highlighting a novel combination approach for evaluation in clinical trials. DisclosuresNo relevant conflicts of interest to declare.

Design, synthesis and in vitro evaluation of stilbene derivatives as novel LSD1 inhibitors for AML therapy.

LSD1 is implicated in a number of malignancies and has emerged as an exciting target. As part of our sustained efforts to develop novel reversible LSD1 inhibitors for epigenetic therapy of cancers, in this study, we reported a series of stilbene derivatives and evaluated their LSD1 inhibitory activities, obtaining several compounds as potent LSD1 inhibitors with IC50 values in submicromolar range. Enzyme kinetics studies and SPR assay suggested that compound 8c, the most active LSD1 inhibitor (IC50 = 283 nM), potently inhibited LSD1 in a reversible and FAD competitive manner. Consistent with the kinetics data, molecular docking showed that compound 8c can be well docked into the FAD binding site of LSD1. Flow cytometry analysis showed that compound 8c was capable of up-regulating the expression of the surrogate cellular biomarker CD86 in THP-1 human leukemia cells, suggesting the ability to block LSD1 activity in cells. Compound 8c showed good inhibition against THP-1 and MOLM-13 cells with IC50 values of 5.76 and 8.34 μM, respectively. Moreover, compound 8c significantly inhibited colony formation of THP-1 cells dose dependently.

Abstract B08: Targeting NOTCH activation in small cell lung cancer through LSD1 inhibition

Abstract Small cell lung cancer (SCLC) is a recalcitrant, lethal tumor type for which little change to first-line standard-of-care treatment has been made over the last few decades. Unlike non-small cell lung cancer (NSCLC), SCLC harbors few actionable mutations for therapeutic intervention. Activation of NOTCH signaling impairs neuroendocrine differentiation and suppresses SCLC, but there are no therapeutic strategies to activate NOTCH in SCLC. LSD1 inhibitors were previously shown to have selective activity in SCLC models, but the underlying mechanism was unknown. Here we demonstrate that exposure to the highly potent and selective LSD1 inhibitor, RG6016/ORY1001, induces NOTCH pathway activation, resulting in the suppression of ASCL1, a regulator of neuroendocrine cell state critical for SCLC oncogenesis. LSD1 binds to the NOTCH1 locus, modulating NOTCH1 expression in SCLC cells. In vivo, sensitivity to LSD1 inhibition in SCLC patient-derived xenograft (PDX) models correlates with the extent of NOTCH pathway activation and repression of a neuroendocrine phenotype. LSD1 inhibition represents a novel therapeutic approach to target the activation of NOTCH and suppression of ASCL1 in SCLC. Citation Format: Arnaud Augert, Emily Eastwood, Ali H. Ibrahim, Nan Wu, Eli Grunblatt, Ryan Basom, Keith D. Eaton, Renato Martins, John Poirier, Charles M. Rudin, Francesca Milletti, Fiona Mack, David MacPherson. Targeting NOTCH activation in small cell lung cancer through LSD1 inhibition [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr B08.

Abstract A169: Combination of TPC-144, a reversible LSD1 inhibitor, and a hypomethylating agent resulted in synergistic antitumor efficacy in preclinical models of AML

Abstract Background: Lysine-specific demethylase 1A (LSD1/KDM1A) is a flavin adenine dinucleotide (FAD)-dependent histone demethylase that specifically modifies histone H3 lysine 4 and lysine 9. LSD1 activity is implicated in the pathogenesis of several human cancers, and recent studies indicated that the inhibition of LSD1 is a promising therapeutic strategy for acute myeloid leukemia (AML). Decitabine and azacitidine, FDA-approved agents for the treatment of AML and myelodysplastic syndrome, alter global DNA methylation by inhibiting DNA methyltransferases (DNMTs), resulting in apoptosis and differentiation. Since it is reported that LSD1 directly demethylates and stabilizes DNMT1 by protection from degradation, we hypothesized that an LSD1 inhibitor could augment anti-AML activity of DNMT inhibitors. Previously, we demonstrated that TPC-144, a novel reversible LSD1 inhibitor, was efficacious in preclinical models of AML. Here, we report the activity of TPC-144 in combination with decitabine in cell-based and in vivo studies. Material and Methods: In vitro studies on growth inhibition and apoptosis were conducted using AML cell lines by measuring cellular ATP content (Cell-Titer Glo, Promega) and sub-G1 population, respectively. For expression changes of surface markers, cells were stained with specific antibodies (BD Pharmingen) and analyzed by flow cytometry. mRNA levels of LSD1 target genes were quantitated by real-time PCR. LINE-1 methylation levels were evaluated in Global DNA Methylation-LINE-1 Kit (Active Motif). In vivo efficacy was evaluated in human AML xenograft models in SCID mice. TPC-144 and decitabine was administered orally or intraperitoneally, respectively. Results: The combination of TPC-144 and decitabine showed synergistic growth inhibition of human AML cells in vitro. TPC-144 enhanced apoptosis induction in combination with low-dose decitabine at the concentrations at which TPC-144 decreased DNMT1 protein levels and LINE-1 methylation levels. Combination treatment strongly increased expression of surface markers of differentiation such as CD86 and CD11b, compared with single-agent treatment. In AML xenograft models, the combination of TPC-144 and decitabine was tolerated without signs of hematopoietic toxicities, and demonstrated curative antitumor efficacy compared with each inhibitor alone in a decitabine-sensitive xenograft model. Importantly, the combination treatment of TPC-144 and decitabine exhibited strong antitumor efficacy even in a decitabine-insensitive AML model. Conclusions: TPC-144, a novel potent and selective reversible LSD1 inhibitor, demonstrated synergistic antitumor efficacy in combination with decitabine in human AML cell lines and xenograft models. This combination was well tolerated and showed strong efficacy not only in decitabine-sensitive but also in decitabine-insensitive AML models. Therefore, combination of TPC-144 and decitabine could enhance their therapeutic value for the treatment of AML patients. Citation Format: Akiko Osada, Aki Kawagishi, Ryo Hatanaka, Takumitsu Machida, Kenjiro Ito, Satoshi Yamashita, Takahiro Ogawa, Tadashi Imaoka, Kenichi Matsuo, Teruhiro Utsugi, Yoshikazu Iwasawa. Combination of TPC-144, a reversible LSD1 inhibitor, and a hypomethylating agent resulted in synergistic antitumor efficacy in preclinical models of AML [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A169.

Abstract 2032: Combination of epigenetic regulation via LSD1 inhibition with signal transduction inhibitors significantly enhances anti-tumor activity in models of hematologic malignancies

Abstract Combinatorial therapeutic strategies have achieved improved response rates and durability of responses in several malignancies either by selectively targeting distinct and non-overlapping oncogenic signaling pathways (e.g. PARP and phosphoinositide 3-kinase (PI3K) inhibition in subsets of breast and ovarian cancers), or alternatively, inhibiting distinct nodal points of regulation in common oncogenic signaling pathways (e.g BRaf and MEK inhibition in subsets of melanoma). Recent data suggest that deregulated epigenetic modifications may be just as significant as genetic mutations in driving cancer development and growth by inhibition of tumor suppressor activity and activation of oncogenic pathways. We therefore hypothesized that an epigenetic regulator could potentiate the efficacy of a protein kinase inhibitor to result in robust tumor growth inhibition. We previously reported that the potent and selective LSD1 inhibitor INCB059872 potently inhibited tumor growth in multiple tumor xenograft models of AML and SCLC as a single agent and in a combination with standard of care of agents. In this study, we explored the anti-tumor effect of combining INCB059872 and various signal transduction pathway inhibitors, including the PIM kinase inhibitor INCB053914, the JAK1/2 inhibitor ruxolitinib, or the PI3K delta-selective inhibitor INCB050465 in models of human hematologic malignancies. Each of these therapeutic combinations significantly inhibited tumor growth in the Molm-16 human AML xenograft model. Mechanistic studies suggested that MYC expression levels were downregulated by these combinations both in vitro and in vivo. Treatment with INCB059872 alone or in combination with signal transduction kinase inhibitors significantly downregulated cytokines levels, particularly IL-10, sCD40L, and MCP-1 in Molm-16 tumors. These data suggest that the combination of an LSD1 inhibitor and signal transduction inhibitor can co-regulate key tumor intrinsic and extrinsic pathways involved in paracrine or autocrine signaling in AML. In addition to the improved efficacy observed in AML models, the combination of INCB059872 with the PI3Kdelta inhibitor INCB050465 enhanced tumor growth inhibition in the Will-2 xenograft model (GCB subtype, double hit lymphoma), whereas the activity of these single agents were modest in this particular subtype of lymphoma. Additional mechanistic studies are ongoing to further understand the molecular bases of these observations. Taken together, these data suggest that targeting distinct epigenetic and oncogenic signaling pathways may potentiate anti-tumor efficacy and overcome intrinsic resistance mechanisms in specific hematologic malignancies. Citation Format: Sang Hyun Lee, Matthew Stubbs, Ashish Juvekar, Melody Diamond, Antony Chadderton, Robert Collins, Xiaoming Wen, Holly Koblish, Chunhong He, Liangxing Wu, Richard Wynn, Andrew Combs, Chu-Biao Xue, Wenqing Yao, Gregory Hollis, Reid Huber, Peggy Scherle, Bruce Ruggeri. Combination of epigenetic regulation via LSD1 inhibition with signal transduction inhibitors significantly enhances anti-tumor activity in models of hematologic malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2032. doi:10.1158/1538-7445.AM2017-2032

Lysine-Specific Demethylase 1 (LSD1) Inhibitor S2101 Induces Autophagy via the AKT/mTOR Pathway in SKOV3 Ovarian Cancer Cells.

BackgroundS2101 is one of the most potent LSD1 inhibitors, which can inhibit ovarian cancer cells viability. This study aimed to detect the mechanism behind the anticancer properties of S2101 in SKOV3 ovarian cells.Material/MethodsCell viability was tested by Cell Counting Kit-8 (CCK-8) assay. Cellular apoptosis and autophagy were evaluated by flow cytometric analysis using Annexin-V/PI staining methods and Green fluorescent protein (GFP)-fused-LC3 (GFP-LC3), respectively. Western blotting was performed for analyzing the Bax, Bcl-2, mTOR, p-mTOR, p62, LC3-I, LC3-II, AKT, and p-AKT protein expression.ResultsOur results show that the proportion of early apoptotic and late apoptotic cells increased significantly for cells treated with S2101 at a concentration of 100 μM for 48 h. Treatment of S2101 in SKOV3 cells resulted in upregulation of Bax and downregulation of Bcl-2 in a time-dependent manner, indicating that S2101 can induce apoptosis in SKOV3. There was a downward trend in the expression of p62 when the SKOV3cells were treated with 100 μm S2101 for 12 h, 24 h and 48 h. The conversion of LC3-I to LC3-II was increased significantly at 24 h and 48 h. Autophagy was induced by S2101 in SKOV3 cells, evidenced by an increase in punctuate localization of GFP-LC3 and a change in expression of autophagy-related proteins.ConclusionsS2101 treatment decreased the levels of phosphorylated AKT and mTOR. S2101 inhibits SKOV3 cells viability and induces apoptosis and autophagy. The AKT/mTOR signaling pathway was found to be affected by S2101.

Design, synthesis and biological evaluation of [1,2,4]triazolo[1,5-a]pyrimidines as potent lysine specific demethylase 1 (LSD1/KDM1A) inhibitors

A new series of [1,2,4]triazolo[1,5-a]pyrimidine-based LSD1 inhibitors were designed, synthesized, and further evaluated for their cytotoxicity against MGC-803, EC109, A549 and PC-9 cells as well as the ability of inhibiting LSD1. Some of these compounds showed potent inhibition toward LSD1 and selectively inhibited growth of A549 and PC-9 cells. Compound 6l potently inhibited growth of PC-9 cells (IC50 = 0.59 μM), about 4-fold more potent than 5-FU. Further SARs studies led to the identification of compounds 6l-m, which had good growth inhibition against all the tested cancer cell lines and were much more potent than 5-FU and GSK2879552. Besides, compounds 5p, 5q and 6i inhibited LSD1 potently (IC50 = 0.154, 1.19 and 0.557 μM, respectively). Docking studies revealed that compound 5p formed arene-H interactions with Val333 and hydrogen bonds with surrounding Ala331, Met332, and Ala539 residues. Compound 5p significantly inhibited migration of A549 and PC-9 cells in a concentration-dependent manner, but had different effect on the expression of E-cadherin and N-cadherin. The [1,2,4]triazolo[1,5-a]pyrimidine scaffold may serve as a starting point for developing potent LSD1 inhibitors for cancer therapy.

C–H activation enables a rapid structure–activity relationship study of arylcyclopropyl amines for potent and selective LSD1 inhibitors

We describe the structure-activity relationship of various arylcyclopropylamines (ACPAs), which are potent LSD1 inhibitors. More than 45 ACPAs were synthesized rapidly by an unconventional method that we have recently developed, consisting of a C-H borylation and cross-coupling sequence starting from cyclopropylamine. We also generated NCD38 derivatives, which are known as LSD1 selective inhibitors, and discovered a more effective inhibitor compared to the original NCD38.

Pyrrole- and indole-containing tranylcypromine derivatives as novel lysine-specific demethylase 1 inhibitors active on cancer cells

A new series of pyrrole/indole-containing tranylcypromine analogues is reported as potent and selective LSD1 inhibitors active in leukemia.

RN-1, a Potent and Selective LSD1 Inhibitor, Induces High Levels of Fetal Hemoglobin (HbF) in Anemic Baboons (P. anubis)

Increased levels of fetal hemoglobin are associated with decreased symptoms and increased life span in patients with sickle cell disease (SCD). Hydroxyurea, the only drug currently approved for SCD, is not effective in a large fraction of patients and therefore new agents are currently needed. Recent evidence has shown that LSD1, an enzyme that removes monomethyl and dimethyl residues from the lys4 residue of histone H3, is a repressor of γ-globin expression. Tranylcypromine (TCP), an LSD1 inhibitor, was shown to increase γ-globin expression in human βYAC transgenic mice (Shi et al Nat Med 19:291, 2013). Because the arrangement and developmental stage-specific expression pattern of the β-like globin genes is highly conserved between man and other simian primates, the use of an simian primate animal model such as the baboon (P.anubis) is the best predictor of the activity of HbF-inducing agents in man. In this investigation we compared the effect of TCP and the more potent and selective LSD1 inhibitor, RN-1, on HbF expression in anemic baboons (P. anubis). In vitro assays have shown that the LSD1 IC50 of RN-1 is at least 1000 fold less than TCP. Animals were phlebotomized for 14d prior to drug treatment to attain an Hct=20 to induce reticulocytosis and establish baseline HbF levels and were maintained at this Hct by periodic phlebotomies during the course of the experiment. In four baboons treated with varying doses of TCP (2-6mg/kg/; 10-20d; sc) low levels of HbF (4.9-7.9% HbF) were induced that were only slightly higher than those observed at the pretreatment baseline (2.2-4.1% HbF). In contrast, treatment with varying doses of RN-1 (2.5-0.125 mg/kg/d; 5-10d) induced high levels of HbF, F reticulocytes, and F cells in 5 of 6 animals (see Table). At high doses of drug the ratio of 5’Iγ/3’Vγ synthesis was >2 demonstrating a near-complete reversion to the pattern of fetal stage expression. Peak levels of F reticulocytes and γ-globin synthesis were observed 8d and HbF and F cells 11d after the first day of drug administration. Increased γ-globin mRNA levels (γ/γ+β) in reticulocytes measured by RT-PCR showed that increased HbF levels were not due to translational effects. Bisulfite sequence analysis showed that levels of DNA methylation of the γ-globin promoter were similar in pre- and post-treatment BM erythroid precursors from two animals. Flow cytometry analysis using anti-α4-integrin and anti-baboon RBC antibodies showed that RN-1 treatment altered terminal BM erythroid differentiation by increasing the proportion of less differentiated precursors, however no changes in MCHC or total hemoglobin synthesis (α/γ+β) were observed. RN-1 treatment was associated with decreased ANC (290-870 X 103/μl nadir) and increased platelets (1092-1445 X 103/μl peak) and monocytes that were likely caused by effects on hematopoietic differentiation. The ANC nadir and peak platelet and monocyte counts were observed between d14-19 and resolved with 2-3 days. Similar changes in ANC and platelets, although not in monocytes, are observed following treatment with decitabine and can be controlled by modification of dose and schedule of administration (Lavelle et al Blood 119:1240, 2012). We conclude that RN-1, a more potent LSD1 inhibitor than TCP, is a powerful HbF-inducing drug with activity similar to decitabine and predict that LSD1 inhibitors may be useful drugs for the treatment of sickle cell disease.TableHbF, F-cells, and F-retics in Baboons Treated with RN-1AnimalRN-1 Dose (mg/kg/d)HbF (%)Globin synthesis (γ/γ+β)F-cells (%)F-retics (%)PrePostPrePostPrePostPrePost85482.5 (4d)5.827.3ND0.7828.559.234.692.385490.5 (5d)2.429.50.060.6819.160.833.997.580000.25 (5d)4.120.60.150.4920.347.045.780.785480.20 (5d)3.720.50.040.5236.954.836.989.280010.125 (5d)1.84.8ND0.0613.521.822.931.785490.125 (10d)3.6160.040.2217.642.721.070.0 DisclosuresNo relevant conflicts of interest to declare.