Lysine-specific Demethylase LSD1 Research Articles

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

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

HAUSP Is a Key Epigenetic Regulator of the Chromatin Effector Proteins.

HAUSP (herpes virus-associated ubiquitin-specific protease), also known as Ubiquitin Specific Protease 7, plays critical roles in cellular processes, such as chromatin biology and epigenetics, through the regulation of different signaling pathways. HAUSP is a main partner of the “Epigenetic Code Replication Machinery,” ECREM, a large protein complex that includes several epigenetic players, such as the ubiquitin-like containing plant homeodomain (PHD) and an interesting new gene (RING), finger domains 1 (UHRF1), as well as DNA methyltransferase 1 (DNMT1), histone deacetylase 1 (HDAC1), histone methyltransferase G9a, and histone acetyltransferase TIP60. Due to its deubiquitinase activity and its ability to team up through direct interactions with several epigenetic regulators, mainly UHRF1, DNMT1, TIP60, the histone lysine methyltransferase EZH2, and the lysine-specific histone demethylase LSD1, HAUSP positions itself at the top of the regulatory hierarchies involved in epigenetic silencing of tumor suppressor genes in cancer. This review highlights the increasing role of HAUSP as an epigenetic master regulator that governs a set of epigenetic players involved in both the maintenance of DNA methylation and histone post-translational modifications.

UM171 Preserves Epigenetic Marks that Are Reduced in Ex Vivo Culture of Human HSCs via Potentiation of the CLR3-KBTBD4 Complex.

Human hematopoietic stem cells (HSCs) exhibit attrition of their self-renewal capacity when cultured exvivo, a process that is partially reversed upon treatment with epigenetic modifiers, most notably inhibitors of histone deacetylases (HDACs) or lysine-specific demethylase LSD1. A recent study showed that the human HSC self-renewal agonist UM171 modulates the CoREST complex, leading to LSD1 degradation, whose inhibition mimics the activity of UM171. The mechanism underlying the UM171-mediated loss of CoREST function remains undetermined. We now report that UM171 potentiates the activity of a CULLIN3-E3 ubiquitin ligase (CRL3) complex whose target specificity is dictated by the poorly characterized Kelch/BTB domain protein KBTBD4. CRL3KBTBD4 targets components of the LSD1/RCOR1 corepressor complex for proteasomal degradation, hence re-establishing H3K4me2 and H3K27ac epigenetic marks, which are rapidly decreased upon exvivo culture of human HSCs.

Phf21b imprints the spatiotemporal epigenetic switch essential for neural stem cell differentiation.

Cerebral cortical development in mammals involves a highly complex and organized set of events including the transition of neural stem and progenitor cells (NSCs) from proliferative to differentiative divisions to generate neurons. Despite progress, the spatiotemporal regulation of this proliferation-differentiation switch during neurogenesis and the upstream epigenetic triggers remain poorly known. Here we report a cortex-specific PHD finger protein, Phf21b, which is highly expressed in the neurogenic phase of cortical development and gets induced as NSCs begin to differentiate. Depletion of Phf21b in vivo inhibited neuronal differentiation as cortical progenitors lacking Phf21b were retained in the proliferative zones and underwent faster cell cycles. Mechanistically, Phf21b targets the regulatory regions of cell cycle promoting genes by virtue of its high affinity for monomethylated H3K4. Subsequently, Phf21b recruits the lysine-specific demethylase Lsd1 and histone deacetylase Hdac2, resulting in the simultaneous removal of monomethylation from H3K4 and acetylation from H3K27, respectively. Intriguingly, mutations in the Phf21b locus associate with depression and mental retardation in humans. Taken together, these findings establish how a precisely timed spatiotemporal expression of Phf21b creates an epigenetic program that triggers neural stem cell differentiation during cortical development.

Long intergenic non-protein-coding RNA 01446 facilitates the proliferation and metastasis of gastric cancer cells through interacting with the histone lysine-specific demethylase LSD1

Growing evidences illustrated that long non-coding RNAs (lncRNAs) exhibited widespread effects on the progression of human cancers via various mechanisms. Long intergenic non-protein-coding RNA 01446 (LINC01446), a 3484-bp ncRNA, is known to locate at chromosome 7p12.1. However, its biological functions and specific action mechanism in gastric cancer (GC) are still unclear. In our study, LINC01446 was proved to be markedly upregulated in GC tissues relative to the normal tissues, and positively correlated with the poor survival of GC patients. The multivariate Cox regression model showed that LINC01446 functioned as an independent prognostic factor for the survival of GC patients. Functionally, LINC01446 facilitated the proliferation and metastasis of GC cells. Moreover, RNA-seq analysis demonstrated that LINC01446 knockdown primarily regulated the genes relating to the growth and migration of GC. Mechanistically, LINC01446 could widely interact with histone lysine-specific demethylase LSD1 and recruit LSD1 to the Ras-related dexamethasone-induced 1 (RASD1) promoter, thereby suppressing RASD1 transcription. Overall, these findings suggest that LINC01446/LSD1/RASD1 regulatory axis may provide bona fide targets for anti-GC therapies.

Inhibition of lysine-specific demethylase LSD1 induces senescence in Glioblastoma cells through a HIF-1α-dependent pathway

Senescence is a stress-responsive cellular program that leads to cell cycle arrest. In cancer cells, senescence has profound implications for tumor aggressiveness and clinical outcome, but the molecular events that provoke cancer cells to undergo senescence remain unclear. Herein, we provide evidence that the histone demethylase LSD1/KDM1A supports the growth of Glioblastoma tumor cells and its inhibition triggers senescence response. LSD1 is a histone modifier that participates in key aspects of gene transcription as well as in the regulation of methylation dynamics of non-histone proteins. We found that down-regulation of LSD1 inhibits Glioblastoma cell growth, impairs mTOR pathway and cell migration and induces senescence. At mechanistic level, we found that LSD1 regulates HIF-1α protein stability. Pharmacological inhibition or siRNA-mediated silencing of LSD1 expression effectively reduces HIF-1α protein levels, which suffices for the induction of senescence. Our findings elucidate a mechanism whereby LSD1 controls senescence in Glioblastoma tumor cells through the regulation of HIF-1α, and we propose the novel defined LSD1/HIF-1α axis as a new target for the therapy of Glioblastoma tumors.

Expanding the Role of the Histone Lysine-Specific Demethylase LSD1 in Cancer.

Studies of alterations in histone methylation in cancer have led to the identification of histone methyltransferases and demethylases as novel targets for therapy. Lysine-specific demethylase 1 (LSD1, also known as KDM1A), demethylates H3K4me1/2, or H3K9me1/2 in a context-dependent manner. In addition to the well-studied role of LSD1 in the epigenetic regulation of histone methylation changes, LSD1 regulates the methylation dynamic of several non-histone proteins and participates in the assembly of different long noncoding RNA (lncRNA_ complexes. LSD1 is highly expressed in various cancers, playing a pivotal role in different cancer-related processes. Here, we summarized recent findings on the role of LSD1 in the regulation of different biological processes in cancer cells through dynamic methylation of non-histone proteins and physical association with dedicated lncRNA.

Long Intergenic Non-Protein Coding RNA 01446 Facilitates the Proliferation and Metastasis of Gastric Cancer Cells Through Interacting with the Histone Lysine-Specific Demethylase LSD1

Long Intergenic Non-Protein Coding RNA 01446 Facilitates the Proliferation and Metastasis of Gastric Cancer Cells Through Interacting with the Histone Lysine-Specific Demethylase LSD1

The lysine-specific histone demethylase LSD1 controls cell cycle progression in liver fibrosis and cancer by alteration of protein expression profiles

The lysine-specific histone demethylase LSD1 controls cell cycle progression in liver fibrosis and cancer by alteration of protein expression profiles

Role of the DNA repair glycosylase OGG1 in the activation of murine splenocytes

OGG1 (8-oxoguanine-DNA glycosylase) is the major DNA repair glycosylase removing the premutagenic DNA base modification 8-oxo-7,8-dihydroguanine (8-oxoG) from the genome of mammalian cells. In addition, there is accumulating evidence that OGG1 and its substrate 8-oxoG might function in the regulation of certain genes, which could account for an attenuated immune response observed in Ogg1−/− mice in several settings. Indications for at least two different mechanisms have been obtained. Thus, OGG1 could either act as an ancillary transcription factor cooperating with the lysine-specific demethylase LSD1 or as an activator of small GTPases. Here, we analysed the activation by lipopolysaccaride (LPS) of primary splenocytes obtained from two different Ogg1−/− mouse strains. We found that the induction of TNF-α expression was reduced in splenocytes (in particular macrophages) of both Ogg1−/− strains. Notably, an inhibitor of LSD1, OG-L002, reduced the induction of TNF-α mRNA in splenocytes from wild-type mice to the level observed in splenocytes from Ogg1−/− mice and had no influence in the latter cells. In contrast, inhibitors of the MAP kinases p38 and JNK as well as the antioxidant N-acetylcysteine attenuated the LPS-stimulated TNF-α expression both in the absence and presence of OGG1. The free base 8-oxo-7,8-dihydroguanine had no influence on the TNF-α expression in the splenocytes. The data demonstrate that OGG1 plays a role in an LSD1-dependent pathway of LPS-induced macrophage activation in mice.

Lysine-specific demethylase LSD1 regulates autophagy in neuroblastoma through SESN2-dependent pathway

Autophagy is a physiological process, important for recycling of macromolecules and maintenance of cellular homeostasis. Defective autophagy is associated with tumorigenesis and has a causative role in chemotherapy resistance in leukemia and in solid cancers. Here, we report that autophagy is regulated by the lysine-specific demethylase LSD1/KDM1A, an epigenetic marker whose overexpression is a feature of malignant neoplasia with an instrumental role in cancer development. In the present study, we determine that two different LSD1 inhibitors (TCP and SP2509) as well as selective ablation of LSD1 expression promote autophagy in neuroblastoma cells. At a mechanistic level, we show that LSD1 binds to the promoter region of Sestrin2 (SESN2), a critical regulator of mTORC1 activity. Pharmacological inhibition of LSD1 triggers SESN2 expression that hampers mTORC1 activity, leading to enhanced autophagy. SESN2 overexpression suffices to promote autophagy in neuroblastoma cells, while loss of SESN2 expression reduces autophagy induced by LSD1 inhibition. Our findings elucidate a mechanism whereby LSD1 controls autophagy in neuroblastoma cells through SESN2 transcription regulation, and we suggest that pharmacological targeting of LSD1 may have effective therapeutic relevance in the control of autophagy in neuroblastoma.

Abstract 3088: Combination of 4SC-202 and IFN-γ restores mature APC phenotype in AML cells

Abstract Acute myeloid leukemia (AML) is characterized by a differentiation block resulting in accumulation of immature myeloid cells. Differentiation therapy, e.g. with all-trans retinoic acid (ATRA) has become a part of standard treatment for the acute promyelocytic leukemia variant of AML. Other agents like cytokines and HDAC inhibitors were shown to enhance differentiation of AML cells, which results in apoptosis induction or re-sensitization to ATRA. However, a differentiation therapy resulting in the restoration of antigen presenting function of AML cells would be a more desirable asset, since such cells will be able to induce or enhance immune response against residual malignant AML cells. Here, we analyzed the impact of 4SC-202 alone and in combination with different cytokines on differentiation of AML cell lines THP-1, HL-60 and MOLM-13 (AML-M4, -M2, and -M5 class, respectively). Myeloid lineage markers CD11b, CD86, CD14 and HLA-DR were analyzed by flow cytometry and quantitative PCR. Cytokine production was measured using Luminex multiplex system. Furthermore, chromatin immunoprecipitation (ChIP) was performed to assess the direct impact of 4SC-202 on promoter histone acetylation of differentiation genes. 4SC-202 is an orally available clinical stage epigenetic small molecule inhibitor which specifically targets histone deacetylases HDAC class I isoenzymes 1-3 as well as the lysine-specific demethylase LSD1 (KDM1A). 4SC-202 treatment of AML cell lines resulted in a dose-dependent increase of myeloid differentiation genes CD86 and ITGAM (=CD11b) on mRNA and protein level. On the chromatin level, 4SC-202 correspondently induced an upregulation of chromatin activation marks like acetylation of histone H3 lysine 9 and 27 (H3K9ac, H3K27ac) at the promoters of these genes. Interestingly, reference compounds like ATRA, bexarotene, GM-CSF and TNF-α were not as efficacious as 4SC-202 in the induction of differentiation markers in AML cells, whereas IFN-γ strongly induced MHC class II molecules HLA-DR and myeloid lineage marker CD14. Finally, the combination of 4SC-202 and IFN-γ resulted in a strong and complementary induction of differentiation and APC markers CD11b, CD14, CD86 and HLA-DR resulting in a CD86high/HLA-DRhigh phenotype which was able to produce pro-inflammatory cytokines. In summary, we demonstrate that 4SC-202 induces differentiation of AML cells of different subtypes and thus provide promising preclinical data for applying this novel epigenetic modulator for differentiation therapy of AML. Moreover, the mature APC phenotype induced by the combination of 4SC-202 and IFN-γ may further suggest an application in consolidation treatment of AML to enhance or even elicit a tumor-specific T-cell response against residual malignant AML cells preventing AML recurrence. Citation Format: Anne Catherine Bretz, Ulrike Parnitzke, Kerstin Kronthaler, Svetlana Hamm. Combination of 4SC-202 and IFN-γ restores mature APC phenotype in AML cells [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 3088. doi:10.1158/1538-7445.AM2017-3088

Abstract 2632: 4SC-202 induces inflamed tumor microenvironment, strongly enhances tumor infiltration with cytotoxic T cells and primes tumors for anti-PD1/PD-L1 therapy

Abstract Various HDAC inhibitors were described as beneficially affecting anti-tumoral immune response. Although different HDAC inhibitors were investigated in syngeneic tumor models, the mode of anti-tumoral action is not yet fully understood. Here, we analyzed the anti-tumoral mode-of-action (MOA) of 4SC-202, an orally available clinical stage epigenetic small molecule inhibitor specifically targeting histone deacetylases HDAC class I as well as the lysine-specific demethylase LSD1. To ensure that the conclusions would be relevant for the clinical situation we used a clinically equivalent dose and schedule. Anti-tumoral efficacy and the impact of 4SC-202 alone or in combination with an anti-PD-L1 antibody on the tumor microenvironment were analyzed in the syngeneic colorectal CT26 model in immunocompetent BALB/c or in nude/irradiated BALB/c mice. A broad spectrum HDAC inhibitor was used for comparison. Transcriptome analysis was performed by RNA-Seq, and the composition of immune cell subpopulations in the tumor was determined by flow cytometry. 4SC-202 treatment at clinically relevant dosage regimen significantly inhibited growth of CT26 tumors. A competent immune system was apparently necessary for the anti-tumoral effect of 4SC-202 since its tumor-reducing effect was lost in immunocompromised mice. 4SC-202 treatment led to an increase of MHC class II molecules and enhanced expression of inflammatory markers like IFN-γ and various chemokines in the tumors. Detailed analysis of the tumor microenvironment revealed that 4SC-202 strongly increased the number of tumoricidal cytotoxic T cells (CTL). In contrast, a broad-spectrum HDAC inhibitor tested in the same model demonstrated anti-tumoral efficacy but did not affect the number of CTLs in tumors demonstrating that HDAC inhibitors employ different MOAs for their anti-tumoral response and that the effect on CTLs is not attributed to HDAC inhibition in general. Since the T cell abundance is pre-requisite for the efficacy of PD1/PD-L1 blockade, we tested the combination of clinically relevant dosage regiment of 4SC-202 with an anti-PD-L1 antibody. The combined treatment was more efficacious than monotherapies and resulted in significantly longer survival. 4SC-202 already demonstrated a favorable safety profile in a phase I clinical trial with relapsed or refractory hematological malignancies with two objective responses (1 CR, 1 PR) and disease stabilizations in several patients. 4SC-202’s immune priming capacity offers further options for clinical development of 4SC-202 in combination with various cancer immunotherapy approaches. Citation Format: Tanja Wulff, Kerstin Kronthaler, Sabine Schrepfer, Ulrike Parnitzke, Anne Catherine Bretz, Roland Baumgartner, Svetlana Hamm. 4SC-202 induces inflamed tumor microenvironment, strongly enhances tumor infiltration with cytotoxic T cells and primes tumors for anti-PD1/PD-L1 therapy [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 2632. doi:10.1158/1538-7445.AM2017-2632

Clinical development of 4SC-202, a combined epigenetic inhibitor of HDAC class I and LSD1, to overcome anti-PD-1 refractoriness and increase efficacy of checkpoint inhibition.

e14096 Background: Checkpoint inhibitor (CI) like anti-PD(L)-1 antibodies are a breakthrough innovation, but a high unmet medical need remains for a large proportion of patients not responding. Low mutational load, microsatellite stability and a non-inflamed tumor microenvironment have been described as negative predictive factors. A promising approach to increase the benefit from anti-PD(L)-1 treatment is to alter the tumor microenvironment to a more immune-inflamed phenotype by combination assets. Epigenetic modulation has been reported as one key determining factor in shaping the immune microenvironment and compounds altering these processes, like histone deacetylases (HDAC) inhibitor in particular offer a promising approach. Methods: We report preclinical results from the small molecule compound 4SC-202, an orally available clinical stage epigenetic inhibitor, specifically targeting HDAC class I and lysine-specific demethylase LSD1. Results: 4SC-202 has shown the ability to elicit a variety of immune modulating effects on tumors and their microenvironment by enhancing the expression of tumor associated antigens as well as MHC and co-stimulatory molecules. Combination treatment of 4SC-202 with CI in syngenic mouse models showed a strong synergistic effect leading to enhanced tumor reduction when compared to 4SC-202 or checkpoint inhibitor treatment alone. Importantly, 4SC-202 strongly increased the number of tumoricidal cytotoxic T cells (CTL) without affecting the viability of activated T cells. Conclusions: Anti-PD(L)-1 refractory/non-responding patients remain a population with a high unmet medical need, increasing in number over time. Advanced melanoma is the model tumor for immunotherapy in general and CI in particular. Based on the strong preclinical rationale, we hypothesize that the addition of 4SC-202 to anti-PD-1 treatment may lead to increased immunogenicity of the tumor, an inflamed tumor microenvironment and subsequent clinical benefit in anti-PD-1 refractory/non-responding melanoma patients. The “SENSITIZE” study is intended to proof this concept in a phase Ib/II clinical trial.

AT-04TOWARDS AN EPIGENETIC THERAPY FOR RHABDOID TUMORS -IN VITRO RESULTS FOR THE EPIGENETIC MODULATORS RESMINOSTAT AND 4SC-202

AT-04. TOWARDS AN EPIGENETIC THERAPY FOR RHABDOID TUMORS -IN VITRO RESULTS FOR THE EPIGENETIC MODULATORS RESMINOSTAT AND 4SC-202 Hella Kohlhof2, Tanja Wulff2, Daniel Vitt2, and Michael Fruehwald1; Children’s Hospital Augsburg, Augsburg, Germany; 4SC AG, Martinsried, Germany Among others histone deactyleases 1 and 2 (HDAC1, 2) are overexpressed across all molecular subgroups of AT/RT. Resminostat is an orally available inhibitor of HDAC classes I and IIb; whereas 4SC-202 is a combined inhibitor of HDAC1/2/3 and the lysine specific demethylase LSD1. Additionally, 4SC-202 inhibits the SMO-independent HH signaling which is often aberrantly activated in AT/RT. Both inhibitors have proven tolerability and efficacy in phase I or II trials in adults. Their toxicity profile appears to be favorable while maintaining targeting efficacy. Proliferation and viability of cell lines G401 (RTK), A204 (MRT) and CHLA-02 (AT/ RT) were evaluated by crystal violet staining and MTT, spheroid and colony formation assays. Expression of genes associated with the phenotype of rhabdoid tumors such as SMARCB1, CDKN1C, MYC, CyclinD1 and HHIP was examined by qRT-PCR. As comparative HDACi Vorinostat, Panobinostat, Entinostat, Mocetinostat and Ricolinostat were tested. Resminostat and 4SC-202 exhibited a dose dependent inhibition of proliferation and viability of RT cells. Inhibition of the capacity for clonogenic growth was demonstrated. Gene expression data are currently evaluated. Rhabdoid tumors as paradigmatic epigenetic malignancies appear to be the optimal foci for drugs targeting the epigenetic scaffolding of tumor cells. The epigenetic modulators Resminostat and 4SC-202 warrant further testing in clinical trials in children with rhabdoid tumors. Likely combinatorial therapy will be needed to prove a significant effect on outcome for affected high-risk patients. Neuro-Oncology 18:iii1–iii6, 2016. doi:10.1093/neuonc/now065.3 #The Author(s) 2016. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Abstract C89: The small molecule inhibitor 4SC-202 controls aberrant HH signaling in cancer

Abstract Introduction: 4SC-202, a clinical stage inhibitor of LSD1 and HDAC1, 2 and 3 was investigated to identify its unique mechanism to target cancer cells by controlling aberrant Hedgehog signaling. Experiments: 4SC-202 is an oral available epigenetic modulator exhibiting a combined inhibition of the lysine specific demethylase LSD1 (KDM1A) and histone deacetylases HDAC1, 2 and 3. 4SC-202's impact on Hedgehog signaling activity was tested in a SMO-dependent setting and in a SMO-independent setting. Therefore, Hedgehog signaling in DAOY cells was either activated by addition of sHH or Smoothened agonist or by stable depletion of SUFU for instrinsic, SMO-independent signaling. Pathway activity was determined by the expression of the effector protein GLI and primary target genes. SMO-independent Hedgehog signaling can be driven as well by growth factors like TGF-ß as demonstrated for the pancreatic cancer cell line PANC1. Therefore, 4SC-202's impact on spheroid formation and protein expression was tested in PANC1 cells. Results: Compared to HDAC inhibitors like Vorinostat, LSD1 inhibitors like OGL-002 and Hedgehog inhibitors targeting SMO like Vismodegib, 4SC-202 was able to inhibit canonical as well as non-canonical GLI-driven Hedgehog signaling. Additionally, 4SC-202 was able to prevent spheroid formation of pancreatic cancer cells and inhibit TGF-ß driven signaling in PANC1 cells. Conclusion: Aberrant activity of the Hedgehog signaling pathway has been implicated in the development, progression and relapse of different cancer entities. Especially the interaction of tumor cells and the microenvironment is driven by SMO-independent Hedgehog signals and therefore Hedgehog inhibitors targeting SMO like Vismodegib were not able to demonstrate clinical benefit in SMO-independent Hedgehog driven cancer types like PDAC. The unique feature of 4SC-202 to control both SMO-dependent and SMO-independent HH signaling provides the opportunity to demonstrate activity in Hedgehog driven entities in further clinical investigations. 4SC-202 was safe and well tolerated in a phase I clinical trial (TOPAS) in patients with advanced hematological diseases. With a disease control rate of 83%, one partial responder treated for 8 months and one complete responder treated for 28 months, patients benefited from 4SC-202 treatment and hints of activity could be demonstrated. Citation Format: Hella Kohlhof, Wolfgang Gruber, Daniel Vitt, Fritz Aberger, Tanja Prenzel. The small molecule inhibitor 4SC-202 controls aberrant HH signaling in cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C89.

Modulation of LSD1 phosphorylation by CK2/WIP1 regulates RNF168-dependent 53BP1 recruitment in response to DNA damage

Proper DNA damage response is essential for the maintenance of genome integrity. The E3 ligase RNF168 deficiency fully prevents both the initial recruitment and retention of 53BP1 at sites of DNA damage. In response to DNA damage, RNF168-dependent recruitment of the lysine-specific demethylase LSD1 to the site of DNA damage promotes local H3K4me2 demethylation and ubiquitination of H2A/H2AX, facilitating 53BP1 recruitment to sites of DNA damage. Alternatively, RNF168-mediated K63-linked ubiquitylation of 53BP1 is required for the initial recruitment of 53BP1 to sites of DNA damage and for its function in repair. We demonstrated here that phosphorylation and dephosphorylation of LSD1 at S131 and S137 was mediated by casein kinase 2 (CK2) and wild-type p53-induced phosphatase 1 (WIP1), respectively. LSD1, RNF168 and 53BP1 interacted with each other directly. CK2-mediated phosphorylation of LSD1 exhibited no impact on its interaction with 53BP1, but promoted its interaction with RNF168 and RNF168-dependent 53BP1 ubiquitination and subsequent recruitment to the DNA damage sites. Furthermore, overexpression of phosphorylation-defective mutants failed to restore LSD1 depletion-induced cellular sensitivity to DNA damage. Taken together, our results suggest that LSD1 phosphorylation modulated by CK2/WIP1 regulates RNF168-dependent 53BP1 recruitment directly in response to DNA damage and cellular sensitivity to DNA damaging agents.

First-in-human study of 4SC-202, a novel oral HDAC inhibitor in advanced hematologic malignancies (TOPAS study).

8559 Background: 4SC-202 is a specific inhibitor of protein deacetylases HDAC1, 2 and 3 and lysine specific demethylase LSD1 (KDM1A). Enzymatic inhibition leads to transcriptional repression of Wnt...

Concerted Hydrogen Atom and Electron Transfer Mechanism for Catalysis by Lysine-Specific Demethylase

We calculate the free energy profile for the postulated hydride transfer reaction mechanism for the catalysis of lysine demethylation by lysine-specific demethylase LSD1. The potential energy surface is obtained by using combined electrostatically embedded multiconfiguration molecular mechanics (EE-MCMM) and single-configuration molecular mechanics (MM). We employ a constant valence bond coupling term to obtain analytical energies and gradients of the EE-MCMM subsystem, which contains 45 quantum mechanics (QM) atoms and which is parametrized with density functional calculations employing specific reaction parameters obtained by matching high-level wave function calculations. In the MM region, we employ the Amber ff03 and TIP3P force fields. The free energy of activation at 300 K is calculated by molecular dynamics (MD) umbrella sampling on a system with 102,090 atoms as the maximum of the free energy profile along the reaction coordinate as obtained by the weighted histogram analysis method with 17 umbrella sampling windows. This yields a free energy of activation of only 10 kcal/mol, showing that the previously postulated direct hydride transfer reaction mechanism is plausible, although we find that it is better interpreted as a concerted transfer of a hydrogen atom and an electron.

Lysine-specific histone demethylase LSD1 and the dynamic control of chromatin

The flavin adenine dinucleotide-dependent amine oxidase LSD1 is the first molecularly defined histone demethylase, which specifically demethylates H3K4me1/me2. The enzyme dynamically controls a large variety of biological processes and is associated with protein complexes controlling transcriptional repression and activation. Molecular analysis of the Drosophila LSD1 homolog revealed new insights into the epigenetic control of heterochromatin formation during early embryogenesis, the establishment of transcriptional gene silencing and the epigenetic mechanisms associated with the maintenance of stem cell identity in primordial germline cells. This review summarizes our recent knowledge about the control of enzymatic activity and molecular function of LSD1 enzyme complexes in different model organisms including Schizosaccharomyces pombe, Drosophila and mammals. Finally, new developments in applied cancer research based on molecular analysis of LSD1 in cancer cells are discussed.