Eric di Luccio

Cancers and the NSD family of histone lysine methyltransferases.

Both genetic and epigenetic alterations are responsible for the stepwise initiation and progression of cancers. Only epigenetic aberrations can be reversible, allowing the malignant cell population to revert to a more benign phenotype. The epigenetic therapy of cancers is emerging as an effective and valuable approach to both the chemotherapy and the chemoprevention of cancer. The utilization of epigenetic targets that include histone methyltransferase (HMTase), Histone deacetylatase, and DNA methyltransferase, are emerging as key therapeutic targets. The nuclear receptor binding SET domain (NSD) protein is a family of three HMTases, NSD1, NSD2/MMSET/WHSC1, and NSD3/WHSC1L1, and plays a critical part in chromatin integrity as evidenced by a growing number of conditions linked to the alterations and/or amplification of NSD1, NSD2, and/or NSD3. NSD1, NSD2 and NSD3 are associated with multiple cancers. The amplification of either NSD1 or NSD2 triggers the cellular transformation and thus is key in the early carcinogenesis events. In most cases, reducing the levels of NSD proteins would suppress cancer growth. NSD1 and NSD2 were isolated as genes linked to developmental diseases, such as Sotos syndrome and Wolf-Hirschhorn syndrome, respectively, implying versatile aspects of the NSD proteins. The NSD pathways, however, are not well understood. It is noteworthy that the NSD family is phylogenetically distinct compared to other known lysine-HMTases, Here, we review the current knowledge on NSD1/NSD2/NSD3 in tumorigenesis and prospect their special value for developing novel anticancer drugs.

Nrf2-mediated induction of phase 2 detoxifying enzymes by glyceollins derived from soybean exposed to Aspergillus sojae

Numerous antioxidants have been reported to cause transcriptional activation of several antioxidant enzymes through binding antioxidant‐response element on their promoter region. We, therefore, attempted to examine whether glyceollins, which share common structural features with many phase 2 enzyme inducers and antioxidant activity, could induce detoxifying/antioxidant enzymes. Glyceollins induced NAD(P)H:quinone oxidoreductase activity in a dose‐dependent manner in both mouse hepatoma Hepa1c1c7 and its mutant BPRc1 cells. The compounds also increased the expression of some representative antioxidant enzymes, such as heme oxygenase 1,gamma‐glutamylcysteine synthase, and glutathione reductase, by promoting nuclear translocation of the NF‐E2‐related factor‐2 (Nrf2). Furthermore, phosphorylation of Akt and antioxidant response element‐mediated reporter gene expression were enhanced by glyceollins but suppressed by LY294002, an inhibitor of phosphoinositide 3‐kinases (PI3K). This suggests that glyceollins may cause Nrf2‐mediated phase 2 enzyme induction through activation of the PI3K signaling pathway as well as interaction with Keap1. Our molecular docking simulations also suggest that the glyceollin isomers tightly bind into the binding pocket around Cys151, preventing Nrf2 from docking to Keap1. In conclusion, the current data suggest that glyceollins induced phase 2 detoxifying enzymes likely through promoting nuclear translocation of Nrf2, which is known to be regulated by phosphorylation of Nrf2 and/or disrupting Keap1‐Nrf2 complex formation.

Structural insights into the regulation and the recognition of histone marks by the SET domain of NSD1

The development of epigenetic therapies fuels cancer hope. DNA-methylation inhibitors, histone-deacetylase and histone-methyltransferase (HMTase) inhibitors are being developed as the utilization of epigenetic targets is emerging as an effective and valuable approach to chemotherapy as well as chemoprevention of cancer. The nuclear receptor binding SET domain (NSD) protein is a family of three HMTases, NSD1, NSD2/MMSET/WHSC1, and NSD3/WHSC1L1 that are critical in maintaining the chromatin integrity. A growing number of studies have reported alterations or amplifications of NSD1, NSD2, or NSD3 in numerous carcinogenic events. Reducing NSDs activity through specific lysine-HMTase inhibitors appears promising to help suppressing cancer growth. However, little is known about the NSD pathways and our understanding of the histone lysine-HMTase mechanism is partial. To shed some light on both the recognition and the regulation of epigenetic marks by the SET domain of the NSD family, we investigate the structural mechanisms of the docking of the histone-H4 tail on the SET domain of NSD1. Our finding exposes a key regulatory and recognition mechanism driven by the flexibility of a loop at the interface of the SET and postSET region. Finally, we prospect the special value of this regulatory region for developing specific and selective NSD inhibitors for the epigenetic therapy of cancers.

In vitro histone lysine methylation by NSD1, NSD2/MMSET/WHSC1 and NSD3/WHSC1L.

BackgroundHistone lysine methylation has a pivotal role in regulating the chromatin. Histone modifiers, including histone methyl transferases (HMTases), have clear roles in human carcinogenesis but the extent of their functions and regulation are not well understood. The NSD family of HMTases comprised of three members (NSD1, NSD2/MMSET/WHSC1, and NSD3/WHSC1L) are oncogenes aberrantly expressed in several cancers, suggesting their potential to serve as novel therapeutic targets. However, the substrate specificity of the NSDs and the molecular mechanism of histones H3 and H4 recognition and methylation have not yet been established.ResultsHerein, we investigated the in vitro mechanisms of histones H3 and H4 recognition and modifications by the catalytic domain of NSD family members. In this study, we quantified in vitro mono-, di- and tri- methylations on H3K4, H3K9, H3K27, H3K36, H3K79, and H4K20 by the carboxyl terminal domain (CTD) of NSD1, NSD2 and NSD3, using histone as substrate. Next, we used a molecular modelling approach and docked 6-mer peptides H3K4 a.a. 1-7; H3K9 a.a. 5-11; H3K27 a.a. 23-29; H3K36 a.a. 32-38; H3K79 a.a. 75-81; H4K20 a.a. 16-22 with the catalytic domain of the NSDs to provide insight into lysine-marks recognition and methylation on histones H3 and H4.ConclusionsOur data highlight the versatility of NSD1, NSD2, and NSD3 for recognizing and methylating several histone lysine marks on histones H3 and H4. Our work provides a basis to design selective and specific NSDs inhibitors. We discuss the relevance of our findings for the development of NSD inhibitors amenable for novel chemotherapies.

Inhibition of Nuclear Receptor Binding SET Domain 2/Multiple Myeloma SET Domain by LEM-06 Implication for Epigenetic Cancer Therapies.

Background: Multiple myeloma SET domain (MMSET)/nuclear receptor binding SET domain 2 (NSD2) is a lysine histone methyltransferase (HMTase) and bona fide oncoprotein found aberrantly expressed in several cancers, suggesting potential role for novel therapeutic strategies. In particular, MMSET/NSD2 is emerging as a target for therapeutic interventions against multiple myeloma, especially t(4;14) myeloma that is associated with a significantly worse prognosis than other biological subgroups. Multiple myeloma is the second most common hematological malignancy in the United States, after non-Hodgkin lymphoma and remains an incurable malignancy. Thus, effective therapeutic strategies are greatly needed. HMTases inhibitors are scarce and no NSDs inhibitors have been isolated.Background: Multiple myeloma SET domain (MMSET)/nuclear receptor binding SET domain 2 (NSD2) is a lysine histone methyltransferase (HMTase) and bona fide oncoprotein found aberrantly expressed in several cancers, suggesting potential role for novel therapeutic strategies. In particular, MMSET/NSD2 is emerging as a target for therapeutic interventions against multiple myeloma, especially t(4;14) myeloma that is associated with a significantly worse prognosis than other biological subgroups. Multiple myeloma is the second most common hematological malignancy in the United States, after non-Hodgkin lymphoma and remains an incurable malignancy. Thus, effective therapeutic strategies are greatly needed. HMTases inhibitors are scarce and no NSDs inhibitors have been isolated. Methods: We used homology modeling, molecular modeling simulations, virtual ligand screening, computational chemistry software for structure-activity relationship and performed in vitro H3K36 histone lysine methylation inhibitory assay using recombinant human NSD2-SET and human H3.1 histone. Results: Here, we report the discovery of LEM-06, a hit small molecule inhibitor of NSD2, with an IC50 of 0.8 mM against H3K36 methylation in vitro. Conclusions: We propose LEM-06 as a hit inhibitor that is useful to further optimize for exploring the biology of NSD2. LEM-06 derivatives may pave the way to specific NSD2 inhibitors suitable for therapeutic efforts against malignancies.

Structure of the thermophilic l-Arabinose isomerase from Geobacillus kaustophilus reveals metal-mediated intersubunit interactions for activity and thermostability.

Thermophilic l-arabinose isomerase (AI), which catalyzes the interconversion of l-arabinose and l-ribulose, can be used to produce d-tagatose, a sugar substitute, from d-galactose. Unlike mesophilic AIs, thermophilic AIs are highly dependent on divalent metal ions for their catalytic activity and thermostability at elevated temperatures. However, the molecular basis underlying the substrate preferences and metal requirements of multimeric AIs remains unclear. Here we report the first crystal structure of the apo and holo forms of thermophilic Geobacillus kaustophilus AI (GKAI) in hexamer form. The structures, including those of GKAI in complex with l-arabitol, and biochemical analyses revealed not only how the substrate-binding site of GKAI is formed through displacement of residues at the intersubunit interface when it is bound to Mn(2+), but also revealed the water-mediated H-bonding networks that contribute to the structural integrity of GKAI during catalysis. These observations suggest metal-mediated isomerization reactions brought about by intersubunit interactions at elevated temperatures are responsible for the distinct active site features that promote the substrate specificity and thermostability of thermophilic AIs.

The structural basis of substrate promiscuity in UDP-hexose 4-epimerase from the hyperthermophilic Eubacterium Thermotoga maritima.

UDP-galactose 4-epimerase (GalE) catalyzes the interconversion of UDP-glucose (UDP-Glc) and UDP-galactose (UDP-Gal), which is a pivotal step in the Leloir pathway for d-galactose metabolism. Although GalE is widely distributed in prokaryotes and eukaryotes, little information is available regarding hyperthermophilic GalE. We overexpressed the TM0509 gene, encoding a putative GalE from Thermotoga maritima (TMGalE), in Escherichia coli and characterized the encoded protein. To further investigate the molecular basis of this enzymes catalytic function, we determined the crystal structures of TMGalE and TMGalE bound to UDP-Glc at resolutions of 1.9 Å and 2.0 Å, respectively. The enzyme was determined to be a homodimer with a molecular mass of 70 kDa. The enzyme could reversibly catalyze the epimerization of UDP-GalNAc/UDP-GlcNAc as well as UDP-Gal/UDP-Glc at elevated temperatures, with an apparent optimal temperature and pH of 80 °C and 7.0, respectively. Our data showed that TM0509 is a UDP-galactosugar 4-epimerase involved in d-galactose metabolism; consequently, this study provides the first detailed characterization of a hyperthermophilic GalE. Moreover, the promiscuous substrate specificity of TMGalE, which is more similar to human GalE than E. coli GalE, supports the notion that TMGalE might exhibit the earliest form of sugar-epimerizing enzymes in the evolution of galactose metabolism.

Abstract 1377: Set7 is a novel histone methyltransferase inSchizosaccharomyces Pombe

Dynamics and plasticity of chromatin regulation is mediated by a molecular ballet of writers, readers and erasers of epigenetic marks on both DNA and histones. The pattern of histone modifications may define a histone code, which is part of intricate networks that ultimately regulate transcriptional events. Dysfunction of histone methylation affects chromatin regulation and is involved in an increasing number of pathologies from cellular transformation to tumor progression and other diseases. However, histone methyltransferase (HMTase) pathways remain to be further explored and better understood. Fission yeast (Schizosaccharomyces pombe) is an ideal model organism to investigate the fundamental mechanisms of chromatin dynamics. Fission yeast possesses 13 SET-containing proteins (Set1 to 13), several of which methylate histones or ribosomes. The catalytic SET domain is highly conserved across eukaryotes (e.g. Set2, a homologue of the oncoprotein NSD2/MMSET/WHSC1 in human). Structure-function studies of HMTases are essential from basic science to translational research. However, full-length structures of fission yeast methyltransferases are still scarce. Here, in a bid to better understand histone methylation and to gain insight for drug-design, we report the identification and structural characterization of a novel histone methyltransferase Set7 (SPCC297.04c), in Schizosaccharomyces Pombe. In this study, we investigated Sp.Set7’s cellular localization and the effect of Sp.Set7 knock-out on the cell cycle and sporulation. Next, we elucidated Sp.Set7’s Lysine substrate specificity on histone H3 and H4 by biochemical assays and mass spectrometry. Finally, we solved the X-ray structure of the apo Sp.Set7 at 2.0 A resolution. In summary, we report the biochemical and structural characterization of a novel histone methyltransferase in fission yeast, which has implications for better understanding the fundamental mechanism of HMTases. Citation Format: Eric Di Luccio, Yunpeng Shen, Damiaan E.H.F Mevius, Yeon Jeong Noh, Jihyeon Kim, Masayo Morishita. Set7 is a novel histone methyltransferase in Schizosaccharomyces Pombe [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 1377. doi:10.1158/1538-7445.AM2017-1377

Abstract 5070: Structure-function studies and drug design on the human histone lysine methyltransferases NSD1, NSD2 and NSD3

Chromatin dynamics are plastic processes controlled by epigenetic modifications on the DNA and histone tails that play a critical role in gene expression. Interestingly, DNA and histones modifications are also under the influence of exogenous factors such as pollutants, smoking, sun exposure, daily diet, alcohol consumption and environmental stress. Departure from “normal” cellular homeostatic conditions is influenced by aberrant accumulation of specific epigenetic patterns, which may contribute to the onset of diseases ranging from diabetes to cancers and neurological disorders. Moreover, epigenetic modifications appear to be inheritable to some extend and affect gene expression in the offspring. One of the key epigenetic modifications is histone methylation, which is catalyzed by histone methyl transferases (HMTases). The nuclear receptor binding SET domain proteins (NSDs) consist of a family of three HMTases, NSD1, NSD2 and NSD3 all of which are bona fide oncoproteins. The NSD HMTases are primarily responsible for methylation of H3K36 and H4K20 in vivo. However, the outlines of the NSDs biological roles in normal and pathological conditions remain unclear. Mutation, alteration or overexpression of the NSD HMTases results in growth defects and is linked to an increasing number of pathologies, carcinogenesis and is a maker for tumor progression and prognosis. However, inhibition of HMTases and especially NSDs by small molecules may offer therapeutic opportunities, especially in cases with poor prognoses, such as multiple myeloma. To date, few HMTase inhibitors exist and no inhibitors specific to the NSD family have been identified. Therefore, a better understanding of the structure-function relationship and the design of specific, selective and bioavailable HMTase inhibitors is essential for novel cancer therapy. In this study, we first we focused on understanding the substrate specificity of the NSDs in vitro to get insight into the druggability of the NSDs. Next, we performed virtual ligand screening and identified the hit molecule LEM-07/14 with an IC50 value of 11.5 µM inhibition against NSD2. LEM-07/14 derivatives resulted in a 45% methylation activity reduction in vitro. Furthermore, we identified BIX-01294 as an NSD inhibitor that differentially inhibits H3K36 methylation by NSD1, NSD2, and NSD3 with IC50 values of 40~112 µM and investigated the molecular basis of inhibition using docking studies on the NSD homology models. Next, we present our X-ray crystallographic efforts on the NSDs and their applications to selective and specific drug-design. Citation Format: Eric Di Luccio, Damiaan E.H.F Mevius, Yunpeng Shen, Yeon Jeong Noh, Jihyeon Kim, Masayo Morishita. Structure-function studies and drug design on the human histone lysine methyltransferases NSD1, NSD2 and NSD3 [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 5070. doi:10.1158/1538-7445.AM2017-5070

Abstract A123: Structure-function studies and drug design on the histone lysine methyl methyltransferases NSD1, NSD2/MMSET/WHSC1, and NSD3/WHSC1L1.

Chromatin remodelers that include histone methyl transferases (HMTases) are becoming a focal point in cancer drug development. The NSD family of three HMTases, NSD1, NSD2/MMSET/WHSC1, and NSD3/WHSC1L are bona fide oncogenes found aberrantly expressed in several cancers, suggesting their potential role for novel therapeutic strategies. Several histone modifiers including HMTase have clear roles in human carcinogenesis but the extent of their functions and regulations are not well understood, especially in pathological conditions. The extents of the NSDs biological roles in normal and pathological conditions remain unclear. In particular, the substrate specificity of the NSDs remains unsettled and discrepant data has been reported. NSD2/MMSET is a focal point for therapeutic interventions against multiple myeloma and especially for t(4;14) myeloma, which is associated with a significantly worse prognosis than other biological subgroups. Multiple myeloma is the second most common hematological malignancy in the United States, after non-Hodgkin lymphoma. Herein, we investigated the in vitro mechanisms of histone mark recognition and modifications by the SET domain of the NSDs and identify novel HMTase inhibitors by virtual ligand screening that may pave the way to specific NSD2/MMSET inhibitors suitable for therapeutic efforts against malignancies that include multiple myeloma. Our data highlights the versatility of NSD1, NSD2, and NSD3 in recognizing and methylating lysine marks on H3 and H4. We report for the discovery of LEM-07 an inhibitor of NSD2/MMSET with an IC50 of 11.2 μM against H3K36 methylation in vitro. LEM-07 would be useful to explore the biology of NSD2. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A123. Citation Format: Eric Di Luccio, Masayo Morishita, Damiaan Mevius, Yunpeng Chen. Structure-function studies and drug design on the histone lysine methyl methyltransferases NSD1, NSD2/MMSET/WHSC1, and NSD3/WHSC1L1. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A123.