Soft drug inhibitors for the epigenetic targets lysine-specific demethylase 1 and histone deacetylases.

Epigenetic modulators such as lysine-specific demethylase 1 (LSD1) and histone deacetylases (HDACs) are drug targets for cancer, neuropsychiatric disease, or inflammation, but inhibitors of these enzymes exhibit considerable side effects. For a potential local treatment with reduced systemic toxicity, we present here soft drug candidates as new LSD1 and HDAC inhibitors. A soft drug is a compound that is degraded in vivo to less active metabolites after having achieved its therapeutic function. This has been successfully applied for corticosteroids in the clinic, but soft drugs targeting epigenetic enzymes are scarce, with the HDAC inhibitor remetinostat being the only example. We have developed new methyl ester-containing inhibitors targeting LSD1 or HDACs and compared the biological activities of these to their respective carboxylic acid cleavage products. In vitro activity assays, cellular experiments, and a stability assay identified potent HDAC and LSD1 soft drug candidates that are superior to their corresponding carboxylic acids in cellular models.

The histone acetyltransferase inhibitor Nir regulates epidermis development.

In addition to its function as an inhibitor of histone acetyltransferases, Nir (Noc2l) binds to p53 and TAp63 to regulate their activity. Here, we show that epidermis-specific ablation of Nir impairs epidermal stratification and barrier function, resulting in perinatal lethality. Nir-deficient epidermis lacks appendages and remains single layered during embryogenesis. Cell proliferation is inhibited, whereas apoptosis and p53 acetylation are increased, indicating that Nir is controlling cell proliferation by limiting p53 acetylation. Transcriptome analysis revealed that Nir regulates the expression of essential factors in epidermis development, such as keratins, integrins and laminins. Furthermore, Nir binds to and controls the expression of p63 and limits H3K18ac at the p63 promoter. Corroborating the stratification defects, asymmetric cell divisions were virtually absent in Nir-deficient mice, suggesting that Nir is required for correct mitotic spindle orientation. In summary, our data define Nir as a key regulator of skin development.

Next-generation sequencing reveals a novel role of lysine-specific demethylase 1 in adhesion of rhabdomyosarcoma cells.

Lysine-specific demethylase 1 (LSD1), a histone lysine demethylase with the main specificity for H3K4me2, has been shown to be overexpressed in rhabdomyosarcoma (RMS) tumor samples. However, its role in RMS biology is not yet well understood. Here, we identified a new role of LSD1 in regulating adhesion of RMS cells. Genetic knockdown of LSD1 profoundly suppressed clonogenic growth in a panel of RMS cell lines, whereas LSD1 proved to be largely dispensable for regulating cell death and short-term survival. Combined RNA and ChIP-sequencing performed to analyze RNA expression and histone methylation at promoter regions revealed a gene set enrichment for adhesion-associated terms upon LSD1 knockdown. Consistently, LSD1 knockdown significantly reduced adhesion to untreated surfaces. Importantly, precoating of the plates with the adhesives collagen I or fibronectin rescued this reduced adhesion of LSD1 knockdown cells back to levels of control cells. Using KEGG pathway analysis, we identified 17 differentially expressed genes (DEGs) in LSD1 knockdown cells related to adhesion processes, which were validated by qRT-PCR. Combining RNA and ChIP-sequencing results revealed that, within this set of genes, SPP1, C3AR1, ITGA10 and SERPINE1 also exhibited increased H3K4me2 levels at their promoter regions in LSD1 knockdown compared to control cells. Indeed, LSD1 ChIP experiments confirmed enrichment of LSD1 at their promoter regions, suggesting a direct transcriptional regulation by LSD1. By identifying a new role of LSD1 in the modulation of cell adhesion and clonogenic growth of RMS cells, these findings highlight the importance of LSD1 in RMS.

Nitroreductase-Mediated Release of Inhibitors of Lysine-Specific Demethylase 1 (LSD1) from Prodrugs in Transfected Acute Myeloid Leukaemia Cells.

Lysine-specific demethylase 1 (LSD1) has evolved as a promising therapeutic target for cancer treatment, especially in acute myeloid leukaemia (AML). To approach the challenge of site-specific LSD1 inhibition, we developed an enzyme-prodrug system with the bacterial nitroreductase NfsB (NTR) that was expressed in the virally transfected AML cell line THP1-NTR+ . The cellular activity of the NTR was proven with a new luminescent NTR probe. We synthesised a diverse set of nitroaromatic prodrugs that by design do not affect LSD1 and are reduced by the NTR to release an active LSD1 inhibitor. The emerging side products were differentially analysed using negative controls, thereby revealing cytotoxic effects. The 2-nitroimidazolyl prodrug of a potent LSD1 inhibitor emerged as one of the best prodrug candidates with a pronounced selectivity window between wild-type and transfected THP1 cells. Our prodrugs are selectively activated and release the LSD1 inhibitor locally, proving their suitability for future targeting approaches.

Lsd1 prevents age-programed loss of beige adipocytes.

Aging is accompanied by major changes in adipose tissue distribution and function. In particular, with time, thermogenic-competent beige adipocytes progressively gain a white adipocyte morphology. However, the mechanisms controlling the age-related transition of beige adipocytes to white adipocytes remain unclear. Lysine-specific demethylase 1 (Lsd1) is an epigenetic eraser enzyme positively regulating differentiation and function of adipocytes. Here we show that Lsd1 levels decrease in aging inguinal white adipose tissue concomitantly with beige fat cell decline. Accordingly, adipocyte-specific increase of Lsd1 expression is sufficient to rescue the age-related transition of beige adipocytes to white adipocytes in vivo, whereas loss of Lsd1 precipitates it. Lsd1 maintains beige adipocytes by controlling the expression of peroxisome proliferator-activated receptor α (Ppara), and treatment with a Ppara agonist is sufficient to rescue the loss of beige adipocytes caused by Lsd1 ablation. In summary, our data provide insights into the mechanism controlling the age-related beige-to-white adipocyte transition and identify Lsd1 as a regulator of beige fat cell maintenance.

Overexpression of histone demethylase Fbxl10 leads to enhanced migration in mouse embryonic fibroblasts.

Cell migration is a central process in the development and maintenance of multicellular organisms. Tissue formation during embryonic development, wound healing, immune responses and invasive tumors all require the orchestrated movement of cells to specific locations. Histone demethylase proteins alter transcription by regulating the chromatin state at specific gene loci. FBXL10 is a conserved and ubiquitously expressed member of the JmjC domain-containing histone demethylase family and is implicated in the demethylation of H3K4me3 and H3K36me2 and thereby removing active chromatin marks. However, the physiological role of FBXL10 in vivo remains largely unknown. Therefore, we established an inducible gain of function model to analyze the role of Fbxl10 and compared wild-type with Fbxl10 overexpressing mouse embryonic fibroblasts (MEFs). Our study shows that overexpression of Fbxl10 in MEFs doesn't influence the proliferation capability but leads to an enhanced migration capacity in comparison to wild-type MEFs. Transcriptome and ChIP-seq experiments demonstrated that Fbxl10 binds to genes involved in migration like Areg, Mdk, Lmnb1, Thbs1, Mgp and Cxcl12. Taken together, our results strongly suggest that Fbxl10 plays a critical role in migration by binding to the promoter region of migration-associated genes and thereby might influences cell behaviour to a possibly more aggressive phenotype.

Lysine Methyltransferase 9 (KMT9) Is an Actionable Target in Muscle-Invasive Bladder Cancer.

Novel treatment modalities are imperative for the challenging management of muscle-invasive and metastatic BC to improve patient survival rates. The recently identified KMT9, an obligate heterodimer composed of KMT9α and KMT9ß, regulates the growth of various types of tumors such as prostate, lung, and colon cancer. While the overexpression of KMT9α was previously observed to be associated with aggressive basal-like MIBC in an analysis of patients' tissue samples, a potential functional role of KMT9 in this type of cancer has not been investigated to date. In this study, we show that KMT9 regulates proliferation, migration, and invasion of various MIBC cell lines with different genetic mutations. KMT9α depletion results in the differential expression of genes regulating the cell cycle, cell adhesion, and migration. Differentially expressed genes include oncogenes such as EGFR and AKT1 as well as mediators of cell adhesion or migration such as DAG1 and ITGA6. Reduced cell proliferation upon KMT9α depletion is also observed in Pten/Trp53 knockout bladder tumor organoids, which cannot be rescued with an enzymatically inactive KMT9α mutant. In accordance with the idea that the catalytic activity of KMT9 is required for the control of cellular processes in MIBC, a recently developed small-molecule inhibitor of KMT9 (KMI169) also impairs cancer cell proliferation. Since KMT9α depletion also restricts the growth of xenografts in mice, our data suggest that KMT9 is an actionable novel therapeutic target for the treatment of MIBC.

Structure-guided design of a selective inhibitor of the methyltransferase KMT9 with cellular activity.

Inhibition of epigenetic regulators by small molecules is an attractive strategy for cancer treatment. Recently, we characterised the role of lysine methyltransferase 9 (KMT9) in prostate, lung, and colon cancer. Our observation that the enzymatic activity was required for tumour cell proliferation identified KMT9 as a potential therapeutic target. Here, we report the development of a potent and selective KMT9 inhibitor (compound 4, KMI169) with cellular activity through structure-based drug design. KMI169 functions as a bi-substrate inhibitor targeting the SAM and substrate binding pockets of KMT9 and exhibits high potency, selectivity, and cellular target engagement. KMT9 inhibition selectively downregulates target genes involved in cell cycle regulation and impairs proliferation of tumours cells including castration- and enzalutamide-resistant prostate cancer cells. KMI169 represents a valuable tool to probe cellular KMT9 functions and paves the way for the development of clinical candidate inhibitors as therapeutic options to treat malignancies such as therapy-resistant prostate cancer.

Impairment of prostate cancer cell growth by a selective and reversible lysine-specific demethylase 1 inhibitor.

Post-translational modifications of histones by chromatin modifying enzymes regulate chromatin structure and gene expression. As deregulation of histone modifications contributes to cancer progression, inhibition of chromatin modifying enzymes such as histone demethylases is an attractive therapeutic strategy to impair cancer growth. Lysine-specific demethylase 1 (LSD1) removes mono- and dimethyl marks from lysine 4 or 9 of histone H3. LSD1 in association with the androgen receptor (AR) controls androgen-dependent gene expression and prostate tumor cell proliferation, thus highlighting LSD1 as a drug target. By combining protein structure similarity clustering and in vitro screening, we identified Namoline, a γ-pyrone, as a novel, selective and reversible LSD1 inhibitor. Namoline blocks LSD1 demethylase activity in vitro and in vivo. Inhibition of LSD1 by Namoline leads to silencing of AR-regulated gene expression and severely impairs androgen-dependent proliferation in vitro and in vivo. Thus, Namoline is a novel promising starting compound for the development of therapeutics to treat androgen-dependent prostate cancer.

KMT9 Controls Stemness and Growth of Colorectal Cancer.

Colorectal cancer is among the leading causes of cancer-associated deaths worldwide. Treatment failure and tumor recurrence due to survival of therapy-resistant cancer stem/initiating cells represent major clinical issues to overcome. In this study, we identified lysine methyltransferase 9 (KMT9), an obligate heterodimer composed of KMT9α and KMT9ß that monomethylates histone H4 at lysine 12 (H4K12me1), as an important regulator in colorectal tumorigenesis. KMT9α and KMT9ß were overexpressed in colorectal cancer and colocalized with H4K12me1 at promoters of target genes involved in the regulation of proliferation. Ablation of KMT9α drastically reduced colorectal tumorigenesis in mice and prevented the growth of murine as well as human patient-derived tumor organoids. Moreover, loss of KMT9α impaired the maintenance and function of colorectal cancer stem/initiating cells and induced apoptosis specifically in this cellular compartment. Together, these data suggest that KMT9 is an important regulator of colorectal carcinogenesis, identifying KMT9 as a promising therapeutic target for the treatment of colorectal cancer. SIGNIFICANCE: The H4K12 methyltransferase KMT9 regulates tumor cell proliferation and stemness in colorectal cancer, indicating that targeting KMT9 could be a useful approach for preventing and treating this disease.

KMT9 monomethylates histone H4 lysine 12 and controls proliferation of prostate cancer cells.

Histone lysine methylation is generally performed by SET domain methyltransferases and regulates chromatin structure and gene expression. Here, we identify human C21orf127 (HEMK2, N6AMT1, PrmC), a member of the seven-ß-strand family of putative methyltransferases, as a novel histone lysine methyltransferase. C21orf127 functions as an obligate heterodimer with TRMT112, writing the methylation mark on lysine 12 of histone H4 (H4K12) in vitro and in vivo. We characterized H4K12 recognition by solving the crystal structure of human C21orf127-TRMT112, hereafter termed 'lysine methyltransferase 9' (KMT9), in complex with S-adenosyl-homocysteine and H4K12me1 peptide. Additional analyses revealed enrichment for KMT9 and H4K12me1 at the promoters of numerous genes encoding cell cycle regulators and control of cell cycle progression by KMT9. Importantly, KMT9 depletion severely affects the proliferation of androgen receptor-dependent, as well as that of castration- and enzalutamide-resistant prostate cancer cells and xenograft tumors. Our data link H4K12 methylation with KMT9-dependent regulation of androgen-independent prostate tumor cell proliferation, thereby providing a promising paradigm for the treatment of castration-resistant prostate cancer.

Lsd1 regulates skeletal muscle regeneration and directs the fate of satellite cells.

Satellite cells are muscle stem cells required for muscle regeneration upon damage. Of note, satellite cells are bipotent and have the capacity to differentiate not only into skeletal myocytes, but also into brown adipocytes. Epigenetic mechanisms regulating fate decision and differentiation of satellite cells during muscle regeneration are not yet fully understood. Here, we show that elevated levels of lysine-specific demethylase 1 (Kdm1a, also known as Lsd1) have a beneficial effect on muscle regeneration and recovery after injury, since Lsd1 directly regulates key myogenic transcription factor genes. Importantly, selective Lsd1 ablation or inhibition in Pax7-positive satellite cells, not only delays muscle regeneration, but changes cell fate towards brown adipocytes. Lsd1 prevents brown adipocyte differentiation of satellite cells by repressing expression of the novel pro-adipogenic transcription factor Glis1. Together, downregulation of Glis1 and upregulation of the muscle-specific transcription program ensure physiological muscle regeneration.

Structure-activity studies on N-Substituted tranylcypromine derivatives lead to selective inhibitors of lysine specific demethylase 1 (LSD1) and potent inducers of leukemic cell differentiation.

FAD-dependent lysine-specific demethylase 1 (LSD1) is overexpressed or deregulated in many cancers such as AML and prostate cancer and hence is a promising anticancer target with first inhibitors in clinical trials. Clinical candidates are N-substituted derivatives of the dual LSD1-/monoamine oxidase-inhibitor tranylcypromine (2-PCPA) with a basic amine function in the N-substituent. These derivatives are selective over monoamine oxidases. So far, only very limited information on structure-activity studies about this important class of LSD1 inhibitors is published in peer reviewed journals. Here, we show that N-substituted 2-PCPA derivatives without a basic function or even a polar group are still potent inhibitors of LSD1 in vitro and effectively inhibit colony formation of leukemic cells in culture. Yet, these lipophilic inhibitors also block the structurally related monoamine oxidases (MAO-A and MAO-B), which may be of interest for the treatment of neurodegenerative disorders, but this property is undesired for applications in cancer treatment. The introduction of a polar, non-basic function led to optimized structures that retain potent LSD1 inhibitors but exhibit selectivity over MAOs and are highly potent in the suppression of colony formation of cultured leukemic cells. Cellular target engagement is shown via a Cellular Thermal Shift Assay (CETSA) for LSD1.

The histone code reader Spin1 controls skeletal muscle development.

While several studies correlated increased expression of the histone code reader Spin1 with tumor formation or growth, little is known about physiological functions of the protein. We generated Spin1M5 mice with ablation of Spin1 in myoblast precursors using the Myf5-Cre deleter strain. Most Spin1M5 mice die shortly after birth displaying severe sarcomere disorganization and necrosis. Surviving Spin1M5 mice are growth-retarded and exhibit the most prominent defects in soleus, tibialis anterior, and diaphragm muscle. Transcriptome analyses of limb muscle at embryonic day (E) 15.5, E16.5, and at three weeks of age provided evidence for aberrant fetal myogenesis and identified deregulated skeletal muscle (SkM) functional networks. Determination of genome-wide chromatin occupancy in primary myoblast revealed direct Spin1 target genes and suggested that deregulated basic helix-loop-helix transcription factor networks account for developmental defects in Spin1M5 fetuses. Furthermore, correlating histological and transcriptome analyses, we show that aberrant expression of titin-associated proteins, abnormal glycogen metabolism, and neuromuscular junction defects contribute to SkM pathology in Spin1M5 mice. Together, we describe the first example of a histone code reader controlling SkM development in mice, which hints at Spin1 as a potential player in human SkM disease.

Inactivation of Lsd1 triggers senescence in trophoblast stem cells by induction of Sirt4.

Coordination of energy metabolism is essential for homeostasis of stem cells, whereas an imbalance in energy homeostasis causes disease and accelerated aging. Here we show that deletion or enzymatic inactivation of lysine-specific demethylase 1 (Lsd1) triggers senescence in trophoblast stem cells (TSCs). Genome-wide transcriptional profiling of TSCs following Lsd1 inhibition shows gene set enrichment of aging and metabolic pathways. Consistently, global metabolomic and phenotypic analyses disclose an unbalanced redox status, decreased glutamine anaplerosis and mitochondrial function. Loss of homeostasis is caused by increased expression of sirtuin 4 (Sirt4), a Lsd1-repressed direct target gene. Accordingly, Sirt4 overexpression in wild-type TSCs recapitulates the senescence phenotype initiated by Lsd1 deletion or inhibition. Inversely, absence of Lsd1 enzymatic activity concomitant with knockdown of Sirt4 reestablishes normal glutamine anaplerosis, redox balance and mitochondrial function. In conclusion, by repression of Sirt4, Lsd1 directs the epigenetic control of TSC immortality via maintenance of metabolic flexibility.

KDM4 Inhibition Targets Breast Cancer Stem-like Cells.

Traditional treatments for breast cancer fail to address therapy-resistant cancer stem-like cells that have been characterized by changes in epigenetic regulators such as the lysine demethylase KDM4. Here, we describe an orally available, selective and potent KDM4 inhibitor (QC6352) with unique preclinical characteristics. To assess the antitumor properties of QC6352, we established a method to isolate and propagate breast cancer stem-like cells (BCSC) from individual triple-negative tumors resected from patients after neoadjuvant chemotherapy. Limiting-dilution orthotopic xenografts of these BCSCs regenerated original patient tumor histology and gene expression. QC6352 blocked BCSC proliferation, sphere formation, and xenograft tumor formation. QC6352 also abrogated expression of EGFR, which drives the growth of therapy-resistant triple-negative breast cancer cells. Our findings validate a unique BCSC culture system for drug screening and offer preclinical proof of concept for KDM4 inhibition as a new strategy to treat triple-negative breast cancer. Cancer Res; 77(21); 5900-12. ©2017 AACR.

Lsd1 Ablation Triggers Metabolic Reprogramming of Brown Adipose Tissue.

Previous work indicated that lysine-specific demethylase 1 (Lsd1) can positively regulate the oxidative and thermogenic capacities of white and beige adipocytes. Here we investigate the role of Lsd1 in brown adipose tissue (BAT) and find that BAT-selective Lsd1 ablation induces a shift from oxidative to glycolytic metabolism. This shift is associated with downregulation of BAT-specific and upregulation of white adipose tissue (WAT)-selective gene expression. This results in the accumulation of di- and triacylglycerides and culminates in a profound whitening of BAT in aged Lsd1-deficient mice. Further studies show that Lsd1 maintains BAT properties via a dual role. It activates BAT-selective gene expression in concert with the transcription factor Nrf1 and represses WAT-selective genes through recruitment of the CoREST complex. In conclusion, our data uncover Lsd1 as a key regulator of gene expression and metabolic function in BAT.

Assembly of methylated KDM1A and CHD1 drives androgen receptor-dependent transcription and translocation.

Prostate cancer evolution is driven by a combination of epigenetic and genetic alterations such as coordinated chromosomal rearrangements, termed chromoplexy. TMPRSS2-ERG gene fusions found in human prostate tumors are a hallmark of chromoplexy. TMPRSS2-ERG fusions have been linked to androgen signaling and depend on androgen receptor (AR)-coupled gene transcription. Here, we show that dimethylation of KDM1A at K114 (to form K114me2) by the histone methyltransferase EHMT2 is a key event controlling androgen-dependent gene transcription and TMPRSS2-ERG fusion. We identified CHD1 as a KDM1A K114me2 reader and characterized the KDM1A K114me2-CHD1 recognition mode by solving the cocrystal structure. Genome-wide analyses revealed chromatin colocalization of KDM1A K114me2, CHD1 and AR in prostate tumor cells. Together, our data link the assembly of methylated KDM1A and CHD1 with AR-dependent transcription and genomic translocations, thereby providing mechanistic insight into the formation of TMPRSS2-ERG gene fusions during prostate-tumor evolution.

The histone code reader SPIN1 controls RET signaling in liposarcoma.

The histone code reader Spindlin1 (SPIN1) has been implicated in tumorigenesis and tumor growth, but the underlying molecular mechanisms remain poorly understood. Here, we show that reducing SPIN1 levels strongly impairs proliferation and increases apoptosis of liposarcoma cells in vitro and in xenograft mouse models. Combining signaling pathway, genome-wide chromatin binding, and transcriptome analyses, we found that SPIN1 directly enhances expression of GDNF, an activator of the RET signaling pathway, in cooperation with the transcription factor MAZ. Accordingly, knockdown of SPIN1 or MAZ results in reduced levels of GDNF and activated RET explaining diminished liposarcoma cell proliferation and survival. In line with these observations, levels of SPIN1, GDNF, activated RET, and MAZ are increased in human liposarcoma compared to normal adipose tissue or lipoma. Importantly, a mutation of SPIN1 within the reader domain interfering with chromatin binding reduces liposarcoma cell proliferation and survival. Together, our data describe a molecular mechanism for SPIN1 function in liposarcoma and suggest that targeting SPIN1 chromatin association with small molecule inhibitors may represent a novel therapeutic strategy.

Selective targeting of the BRG/PB1 bromodomains impairs embryonic and trophoblast stem cell maintenance.

Mammalian SWI/SNF [also called Brg/Brahma-associated factors (BAFs)] are evolutionarily conserved chromatin-remodeling complexes regulating gene transcription programs during development and stem cell differentiation. BAF complexes contain an ATP (adenosine 5'-triphosphate)-driven remodeling enzyme (either BRG1 or BRM) and multiple protein interaction domains including bromodomains, an evolutionary conserved acetyl lysine-dependent protein interaction motif that recruits transcriptional regulators to acetylated chromatin. We report a potent and cell active protein interaction inhibitor, PFI-3, that selectively binds to essential BAF bromodomains. The high specificity of PFI-3 was achieved on the basis of a novel binding mode of a salicylic acid head group that led to the replacement of water molecules typically maintained in other bromodomain inhibitor complexes. We show that exposure of embryonic stem cells to PFI-3 led to deprivation of stemness and deregulated lineage specification. Furthermore, differentiation of trophoblast stem cells in the presence of PFI-3 was markedly enhanced. The data present a key function of BAF bromodomains in stem cell maintenance and differentiation, introducing a novel versatile chemical probe for studies on acetylation-dependent cellular processes controlled by BAF remodeling complexes.