Masayo Morishita

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.

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.

Phosphate-Induced Apoptosis in Human Peritoneal Mesothelial Cells in vitro

Background: It has been demonstrated that phosphate uptake through the type III sodium-dependent phosphate co-transporter, Pit-1, induced apoptosis of aortic vascular smooth muscle cells and endothelial cells in vitro. However, the apoptotic effects of high phosphate (HP) level in human peritoneal mesothelial cells (HPMCs) are not known. Methods: To examine whether Pit-1 is expressed in HPMCs, we checked the Western blot assay of immunoreactive Pit-1 and the transcription of Pit-1 by reverse transcriptase PCR. We treated several different phosphate concentrations (1–4 mM) and calcium concentrations (1.8 and 2.8 mM) on HPMCs to assess the effects of concentration. MTT, TUNEL assays, and flow cytometry analysis using Annexin V and propidium iodide were performed to identify cell death and apoptosis. Bax and Bcl-2 by Western blot and caspase-3 activity were evaluated by colorimetric assay. In addition, phosphonoformic acid (PFA) and pan-caspase inhibitor, Z-VAD-FMK, were given to prevent phosphate-induced apoptosis. Results: Pit-1 expression on HPMCs was demonstrated. Apoptosis in HPMCs significantly increased with a high concentration of phosphate in a dose- and time-dependent manner, and was enhanced in the presence of 2.8 mM calcium. HP concentrations significantly decreased the anti-apoptotic Bcl-2/Bax ratio and increased caspase-3 activity. The treatment with PFA and Z-VAD-FMK prevented cell death by HP. Conclusion: Phosphate uptake through Pit-1 induces apoptosis in HPMCs by a caspase-related mechanism.

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.

Abstract 676: Substrate specificity and inhibitors of histone lysine HMTase NSD1, NSD2/MMSET/WHSC1, and NSD3/WHSC1L1.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC The nuclear receptor binding SET domain proteins (NSDs) is a family of three histone-lysine N-methyltransferases, NSD1, NSD2/MMSET/WHSC1, and NSD3/WHSC1L1. The NSDs are oncogenes. The amplification of NSD1 has been reported in multiple myeloma, lung cancer, neuroblastomas and glioblastomas. The amplification of either NSD1 or NSD2 triggers the cellular transformation to cancer formation. NSD2 is associated with tumor aggressiveness or prognosis in most types of cancers including prostate cancer and multiple myeloma and is overexpressed in at least 15 different cancers. Increased NSD2 activity was reported in tumor proliferation in glioblastoma multiforms and myeloma, resulting in aberrantly high global levels of H3K36me2. NSD3 is involved in lung cancer and found amplified in breast cancer cell lines and primary breast carcinomas. The abnormal NSD1-NUP98 and NSD3-NUP98 fusions increase H3K36 methylation, leading to acute myeloid leukemia. Although the NSD proteins are instrumental in the development of numerous cancers, their mechanisms of action in this context remain unclear. However, it appears that consequently increased H3K36 methylation by deregulated NSDs concomitant with decreased H3K27 methylation is perhaps one of the underlying mechanisms that alter gene expression profiles. In this study, we defined the molecular determinants of the recognition of histone marks by the NSDs to provide medicinal chemistry insights into the design of selective and specific NSDs inhibitors. Our data highlights the catalytic versatility of NSD1, NSD2, and NSD3 in regulating the epigenome. Our results propose the first molecular models of the binding of 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. Exploiting this data, we derived a virtual ligand screening strategy to identify inhibitors targeting NSD1, NSD2 and NSD3. In vitro, using recombinant NSDs-SET purified enzyme, we performed H3K36me1 inhibitory assays with the top candidates. Here for the first time, we report the discovery of a small subset of NSD inhibitors (namely LEM-xx) that do not violate the empirical Lipinskis rule of five, have a wcLogP < 3 and a total polar surface area <90 A**2. Citation Format: Eric Di Luccio, Masayo Morishita. Substrate specificity and inhibitors of histone lysine HMTase NSD1, NSD2/MMSET/WHSC1, and NSD3/WHSC1L1. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 676. doi:10.1158/1538-7445.AM2013-676 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Abstract 1060: Structural insights into the regulation and the recognition of histone marks by the SET domain of histone lysine HMTase NSD1, NSD2/MMSET/WHSC1, and NSD3/WHSC1L1

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Background: The nuclear receptor binding SET domain (NSD) protein is a family of three histone-lysine N-methyltransferase (HMTase), NSD1, NSD2/MMSET/WHSC1, and NSD3/WHSC1L1 that are critical in maintaining the chromatin integrity. NSD1 methylates H3K36 and H4K20 and is associated with acute myeloid leukemia, multiple myeloma, and lung cancer. The NSD1-NUP98 translocation plays a significant role in childhood acute myeloid leukemia with NUP98-NSD1 being an active H3K36 methylase. NSD1 is amplified in multiple myeloma, lung cancer, neuroblastomas and glioblastomas. NSD2 methylates H3K36 and is linked to prostate cancer and multiple myeloma. Over expression of NSD2 in myeloma cells leads to aberrantly high levels of H3K36 di-methylation, accompanied by a decrease in H3K27 methylation. NSD2 is found over expressed in fifteen different cancers and is associated with tumor aggressiveness or prognosis in most types of cancers. NSD3 methylates H3K36 and is associated with both lung and breast cancers along with the acute myeloid leukemia. The amplification of either NSD1 or NSD2 triggers the cellular transformation. NSD3 is found amplified in breast cancer cell lines and primary breast carcinomas. Reducing NSDs activity through specific and selective lysine-HMTase inhibitors appears promising to help suppressing cancer growth. Little is known about the NSD pathways and our understanding of the histone Lysine-HMTase mechanism is partial. The SET domain of NSD1 has specific mechanisms to recognize histone marks unlike other HMTase. The precise catalytic activities of the NSDs are obscure and discrepancies exist hindering progress in understanding their exact biological functions and pathways in cancer pathogenesis. In this study, we explored the in vitro catalytic activities on histone substrates to understand the substrate recognition and to pave the way for the design of selective and specific NSD inhibitors usable in cancer therapies. Methods: We used both biochemical and computational methods to understand the substrates recognition by the NSDs and to investigate the structural mechanisms happening in the SET domain during the binding of histone tails. Results: A key regulatory and a recognition mechanism is driven by the flexibility of a loop at the interface of the SET and postSET region who rotates ∼45° and translated 7A opening the SET domain for the binding of the peptide ligand. This regulatory loop acts as a seat belt for the ligand and contributes to the discrimination and the substrate specificity. In vitro, The SET domain of the NSDs favor H3 recognition and are able to methylate a range of substrate. To reconcile with the in vivo activities previously reported on H3K36 and H4K20, we propose a cross-talk mechanism controlling the substrate recognition. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1060. doi:1538-7445.AM2012-1060

Abstract B81: Structural insights into the regulation and the recognition of histone marks by the SET domain of NSD1.

Background: The nuclear receptor binding SET domain (NSD) protein is a family of three histone-lysine N-methyltransferase (HMTase), NSD1, NSD2/MMSET/WHSC1, and NSD3/WHSC1L1 that are critical in maintaining the chromatin integrity. NSD1 methylates H3K36 and H4K20 and is associated with acute myeloid leukemia, multiple myeloma, and lung cancer. The NSD1-NUP98 translocation plays a significant role in childhood acute myeloid leukemia with the NUP98-NSD1 fusion protein being an active H3K36 methylase. NSD1 is amplified in multiple myeloma, lung cancer, neuroblastomas and glioblastomas. NSD2 methylates H3K4 and H4K20 and is linked to prostate cancer and multiple myeloma. Increased NSD2 activity was reported in the tumor proliferation in glioblastoma multiform. Over expression of NSD2 in myeloma cells leads to aberrantly high global levels of H3K36 di-methylation, accompanied by a decrease in H3K27 methylation. In myeloma cells, NSD2 contributes to disrupt the chromatin structure and function contributing to the cellular transformation. In addition, NSD2 is found over expressed in fifteen different cancers and is associated with tumor aggressiveness or prognosis in most types of cancers. NSD3 methylates H3K36 and is associated with both lung and breast cancer along with the acute myeloid leukemia. Furthermore, the amplification of either NSD1 or NSD2 triggers the cellular transformation, initiating carcinogenesis events NSD3 is found amplified in breast cancer cell lines and primary breast carcinomas. Reducing NSDs activity through specific and selective 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. Analysis of the recent crystal-structure of the peptide-less SET domain of NSD1 revealed that in absence of ligand, the histone-binding site is occluded preventing any access to the catalytic groove. Therefore, we hypothesized that the SET domain of NSD1 has specific mechanisms to recognize histone marks and to grant access to the histone-binding site, unlike other HMTase. Methods: We used computational methods to investigate the structural mechanisms happening in the SET domain during the binding of the H4-histone tail, exploiting the recent crystal structure of the peptide-less SET domain of NSD1. Results: Our finding exposes a key regulatory and a recognition mechanism driven by the flexibility of a loop at the interface of the SET and postSET region who rotates ∼45° and translated 7 angstroms opening the SET domain for the binding of the peptide ligand. This regulatory loop acts as a seat belt for the ligand and contributes to the discrimination and the substrate specificity. HMTase inhibitors are scare, but their design relies on exploiting dissimilarities in the histone-tail binding pocket. Our data bring significant insight into the design of specific and selective NSD-HMTase inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B81.