Small Molecule Inhibitors of the Human Histone Lysine Methyltransferase NSD2 / WHSC1 / MMSET Identified from a Quantitative High-Throughput Screen with Nucleosome Substrate

The activity of the histone lysine methyltransferase NSD2 is thought to play a driving role in oncogenesis. Both overexpression of NSD2 and point mutations that increase its catalytic activity are associated with a variety of human cancers. While NSD2 is an attractive therapeutic target, no potent, selective and cell-active inhibitors have been reported to date, possibly due to the challenging nature of developing high-throughput assays for NSD2. To establish a platform for the discovery and development of selective NSD2 inhibitors, multiple assays were optimized and implemented. Quantitative high-throughput screening was performed with full-length wild-type NSD2 and a nucleosome substrate against a diverse collection of known bioactives comprising 16,251 compounds. Actives from the primary screen were further interrogated with orthogonal and counter assays, as well as activity assays with the clinically relevant NSD2 mutants E1099K and T1150A. Five confirmed inhibitors were selected for follow-up, which included a radiolabeled validation assay, surface plasmon resonance studies, methyltransferase profiling, and histone methylation in cells. The identification of NSD2 inhibitors that bind the catalytic SET domain and demonstrate activity in cells validates the workflow, providing a template for identifying selective NSD2 inhibitors.

Notably, NSD2 is among the most frequently mutated genes in pediatric cancer genomes (29). The NSD2 SET domain mutation, E1099K, was identified in both acute lymphoblastic leukemia tumors and cell lines with increased H3K36me2 that lack the t(4;14) translocation (21,30). Sequence results of >1000 pediatric cancer genomes, representing 21 different cancers, revealed the E1099K mutation in 14% of t(12;21) ETV6-RUNX1 containing ALLs (21). NSD2 is also among the most frequently mutated genes found in mantle cell lymphoma tumors, where both E1099K and T1150A mutations are observed (31). The E1099K mutation has also been reported in chronic lymphocytic leukemia (CLL), lung and stomach cancers (32)(33)(34)(35). Recombinant NSD2 E1099K showed higher in vitro activity compared to the wild-type enzyme (21). Ectopic expression of NSD2 E1099K induced H3K36me2 and promoted transformation, while knockdown of the mutant enzyme reduced cell line proliferation and tumorigenesis (21).
A major challenge in screening for small molecule inhibitors is that native NSD2 requires nucleosomes as a substrate (17). Recombinant NSD2 does not act on peptides and is thus not amenable to the commonly adapted histone-derived peptide screening platforms. Here we report biochemical assay development and pilot-scale screening using full-length recombinant wildtype, E1099K, and T1150A NSD2 enzymes with purified HeLa nucleosomes as a substrate. Chemical libraries were screened in 3-dose point quantitative high-throughput screening (qHTS) format (43) with the Methyltransferase-Glo (MTase-Glo™) methyltransferase bioluminescence assay (44). A number of counter and orthogonal assays were also developed to characterize the hits from the primary screen. Confirmed hits were validated with a radiolabeled SAM substratebased NSD2 activity assay and further interrogated with binding studies by surface plasmon resonance (SPR), methyltransferase profiling, and activity assessments in U-2 OS cells.

Primary assay development and optimization:
To identify small molecule inhibitors of the wild-type NSD2, the MTase-Glo assay was adapted and optimized for use as a primary assay, with whole nucleosomes as the substrate, in 1,536-well format with 4 µL reaction volumes (Fig. 1B). The MTase-Glo assay reagent measures methyltransferase activity through the coupling of the NSD2 reaction product, S-adenosyl-Lhomocysteine (SAH) to a bioluminescent signal (44). The assay was further optimized for the NSD2 mutants E1099K (Fig. 1C) and T1150A (Fig. 1D), to enable insight into cross-inhibition of clinically-relevant NSD2 enzymes. The enzyme concentrations were optimized to allow a robust signal-to-background ratio (>3.0) in a 15-min reaction at room temperature while consuming ≤20% substrate. The three optimized assays were robust with Z'-values near 0.9. There was no reduction in assay performance in the presence of up to 1.7% DMSO, which represents a triple aliquot of fixed volume (23 nL) transferred via a Kalypsys pintool equipped with a 1,536-pin array (Fig. 1E) (45). A titration of wild-type NSD2 demonstrated a linear correlation with methyltransferase activity as expected (Fig. 1F). between the wild-type NSD2 enzyme concentration and methyltransferase activity (mean ± SD; n = 8). Table 1. Optimized protocol for the NSD2 MTase-Glo UltraGlo luciferase qHTS assay.

Quantitative high-throughput screen (qHTS) for NSD2 inhibitors:
The primary screen was carried out by performing qHTS against 16,251 compounds at 3 concentrations (115 µM, 57.5 µM and 11.5 µM) in 1,536-well plates (Table 1). Commerciallyavailable libraries screened included the LOPAC (1,280 compounds), Prestwick (1,360 compounds), MicroSource (2,000 compounds), and Tocris (1,304 compounds) collections. Additionally, four NCATS libraries were screened, including an epigenetics-focused collection (284 compounds), a natural products library (2,108 compounds), the NPACT library (5,099 compounds) and the NCATS Pharmaceutical Collection (2,816 compounds) (46). These libraries, enriched with pharmacologically active compounds, were selected to evaluate the suitability of the primary and secondary assays to identify inhibitors of NSD2. The overall quality of the primary screen data from 43 plates was high, with average values for Z'-factor of 0.92 ± 0.02, signal-to-background (S/B) of 3.28 ± 0.31, and coefficient of variation (CV) for the DMSO control of 1.68% ± 0.49 ( Fig. 2 A-C). The screen was conducted over a period of four months, so these data demonstrate excellent day-to-day reproducibility. From the initial hit list, 289 compounds were selected as cherry picks and further evaluated in 11-point dose response. Of the 289 cherry picks, the activities of 48 were confirmed against NSD2 with the primary and orthogonal assays with no activity observed by the counter assays. The majority of these compounds were also active against the NSD2 mutants E1099K (45) and T1150A (44).

Hit confirmation and secondary screening:
Of the 16,251 compounds evaluated in the primary screen, 536 compounds were active with a maximum response ≥ 50%, corresponding to a hit rate of 3.3% (Fig. 2D). Promiscuity scores were calculated for each hit and any compounds with a promiscuity score higher than 0.2 were eliminated from the cherry-picking. Next the remaining hits were evaluated by a structural filter to eliminate the pan-assay interference compounds (PAINS) (47). Finally, 289 hit molecules were selected as cherry picks and prepared in 11-point concentration series from library stock solutions for further studies (Fig. 2E).
First, 174 of the cherry picks were confirmed as active in the primary assay (a 60% confirmation rate). Actives were defined as having concentration-response curves (CRCs) in the classes of 1.1, 1.2, 2.1, 2.2 and 3. In brief, classes 1.1 and 1.2 are the highest-confidence complete CRCs containing both upper and lower asymptotes with efficacies > 80% and ≤ 80%, respectively. Classes 2.1 and 2.2 are incomplete CRCs having only one asymptote with efficacies > 80% and < 80%, respectively. Class 3 CRCs show activity at only the highest concentration or are poorly fit. Class 4 CRCs are inactive, having a curve-fit of insufficient efficacy or lacking a fit altogether (43). Secondly, a MTase-Glo counter screen was implemented without NSD2 but containing 200 nM SAH (mimicking 20% substrate conversion), which identified 37 assay interference compounds that might act by inhibiting the coupling enzymes, luciferase or the luminescent signal. Potential redox cycling by compounds was assessed with an Amplex Red assay performed in the presence of reducing agents (48) and 63 compounds were found to be active with a threshold of 3σ. To provide additional evidence for on-target activity against NSD2, the orthogonal EPIgeneous Homogeneous Time-Resolved Fluorescence (HTRF) Methyltransferase Assay was utilized with reaction conditions identical to the primary assay (Fig. 3). The assay measures the NSD2 reaction product SAH, which competitively displaces d2labeled SAH that is pre-bound to anti-SAH labeled with Lumi4-Tb, resulting in a loss of FRET signal (49). Among the 37 compounds found to be active with the MTase-Glo counter assay was a known luciferase inhibitor NCGC00183809 (Fig. 4A). The compound shares structural commonality with the firefly luciferase inhibitor PTC-124 (44,50,51). As such it was likely a false-positive hit that inhibits the UltraGlo luciferase enzyme utilized in the MTase-Glo assay. This notion was supported by the results of the orthogonal assay, which did not indicate inhibition of NSD2 activity. UltraGlo luciferase is a genetically evolved firefly luciferase containing 70 mutations to improve its robustness, thermal stability, and resistance to interference compounds (52,53), which is consistent with the 158-fold reduced potency of NCGC00183809 to UltraGlo compared with firefly luciferase (Fig. 4A). The nonspecific methyltransferase inhibitor DZNep (3deazaneplanocin A) showed similar activities in both the MTase-Glo primary and counter assays; however, no inhibition of NSD2 activity was observed with the orthogonal assay (Fig. 4B).
After the orthogonal assay, 48 confirmed hits remained that were not active in the two counter screens (Fig. 2E). Of the 48 confirmed inhibitors, 45 were also active against the E1099K enzyme, and 44 showed activity against the T1150A mutant. One of these hits is the nonspecific histone lysine methyltransferase inhibitor chaetocin, which was reported to inhibit NSD1-3 (39).
Chaetocin inhibited both wild-type and mutant NSD2 enzymes without showing activity by the MTase-Glo counter assay or Amplex Red assay ( Fig 4C). The hit DA3003-1 is known to be redox active (48,54), and this was corroborated by the Amplex Red counter screen ( Fig 4D). Redox activity is undesirable, because it can result in nonspecific modulation of proteins, activation of cell pathways with redox-switches and cytotoxicity (55). DA3003-1 was nevertheless selected for follow-up studies due to its submicromolar potency with the primary assay (IC 50 = 0.9 µM). In addition to chaetocin and DA3003-1, three other hits were selected for further studies that have not been previously linked to NSD2 inhibition: PF-03882845, TC LPA5 4, and ABT-199 (Table 2). In anticipation of a full-fledged HTS, these five compounds were selected to validate our post-HTS workflow, which is intended to further evaluate compound activities.

Potency evaluation with the HotSpot radiolabel assay:
Biochemical activities of the five selected hit compounds against wild-type and mutant NSD2 enzymes were further validated by the radioisotope-based HotSpot assay (Fig. 5). The HotSpot assay incorporates [ 3 H]SAM to assess total histone methylation by direct measurement of the filter-bound tritiated substrate, without the need for coupling enzymes or antibodies (38). Both DA3003-1 and chaetocin inhibited wild-type and mutant NSD2 enzymes with submicromolar potencies, whereas PF-03882845, TC LPA5 4, and ABT-199 inhibited the enzymes at lowmicromolar concentrations ( Table 2). While chaetocin is known to inhibit NSD2, inhibition by DA3003-1, PF-03882845, TC LPA5 4 and ABT-199 has not been reported. Notably, the HotSpot assay was consistently more sensitive than the MTase-Glo and HTRF assays for both wild-type and mutant enzymes ( Table 2). Overall the reaction conditions were very similar. The MTase-Glo and HTRF assays utilized 8 nM NSD2, 500 nM nucleosomes, 50mM Tris-HCl, pH 8.8, 5mM MgCl 2 , 50mM NaCl, 1mM TCEP, 0.01% Tween; whereas the HotSpot assay used 10 nM NSD2, 400 nM nucleosomes, 50 mM Tris-HCl, pH 8.5, 5 mM MgCl 2 , 50 mM NaCl, 1 mM DTT, 0.01% Brij35. A comparison of IC 50 values determined by the HotSpot assay for all five compounds from reactions containing either 1 mM TCEP or DTT did not suggest that potency differences were due to the reducing agent (data not shown). Each of the five compounds inhibited wild-type, E1099K, and T1150A NSD2 enzymes with similar potencies (Table 2).  All values are derived from one experiment unless followed by a symbol, which case the value represents a global fit to n technical replicate experiments (*n = 2, Ω n = 3, ᶲn = 4, ᶷn = 5).

Direct binding of inhibitors to the NSD2 SET domain:
To support an on-target mechanism of action for the five inhibitors, surface plasmon resonance (SPR) was used to determine whether each inhibitor interacts with the catalytic SET domain of NSD2. As expected the two positive controls, cofactor SAM and product SAH, both bound the NSD2 SET domain with low micromolar affinities (Table 3). With the exception of chaetocin, the inhibitors bound the NSD2 SET domain stoichiometrically, with dissociation constants (K d ) comparable to the in vitro IC 50 values determined by the HotSpot assay (Table 2). Chaetocin bound the SET domain with an apparent dissociation constant of 20 nM. Additionally, the data indicated super-stoichiometric binding, which might be due to a binding ratio higher than 1:1 that is consistent with chaetocin's two disulfide moieties forming adducts with the NSD2 protein, or compound aggregation (data not shown) (56). These data indicate that all five compounds might mediate inhibition of NSD2 by directly binding to the catalytic SET domain.   Table 2.

Inhibitor activities in a cell-based assay:
To evaluate the activities of the five NSD2 inhibitors in cells, U-2 OS human osteosarcoma cells were chosen due to a relatively high expression of endogenous NSD2 protein (57). The cells were treated with each of the five compounds in 10-point dose response from 0.0025 -50 µM for 96 hours. NSD2 inhibition should result in reduced H3K36 di-methylation. Total histone H3 and H3K36me2 levels were measured by western analysis. The positive control DZNep was tested at a concentration of 10 µM in parallel with each compound and also evaluated in dose-response (Fig. 6A). Densitometry was used to quantify both H3K36me2 and total H3 and the densities of H3K36me2 were normalized to those of H3 (Fig. 6B). The growth of U-2 OS cells over 96 hours in the presence of test compounds at the same concentrations was also evaluated (Fig. 6C). The IC 50 value of DZNep for reducing H3K36me2 levels in U-2 OS cells was 390 nM with a modest dose-dependent reduction in U-2 OS confluency (AC 50 = 180 nM). Similar to DZNep, DA3003-1 treatment also resulted in a dose-dependent reduction in H3K36me2; however, higher drug concentrations resulted in cytotoxicity (CC 50 = 270 nM) similar to chaetocin and the control bortezomib, both of which were cytotoxic at all concentrations tested. PF-03882845 reduced H3K36me2 (IC 50 = 3.2 µM) over a range of concentrations that had negligible influence on growth; however, significant cell death at concentrations above 16.7 µM was observed during the western analysis experiment (Fig. 6A), so data from the two highest concentrations were excluded from the potency calculation. TC LPA5 4 did not reduce H3K36me2 over the concentrations tested and minimal reduction in growth was observed. ABT-199 did not appear to reduce H3K36me2 levels, however, concentrations above 17 µM were cytotoxic.

Discussion
A number of histone lysine methyltransferases have been implicated as attractive therapeutic targets in the field of oncology and small molecule inhibitors are in different stages of preclinical and clinical development (58,59). Selective inhibitors of NSD2 are of major interest both to advance basic research and for therapeutic development. However, NSD2 is regarded as a challenging target (60) and no selective inhibitors of NSD2 have been reported to date. Challenges in studying NSD2 in vitro include that the target of NSD2 methylation depends on the nature of the substrate (17) and full-length NSD2 is only active against a nucleosome substrate (38), which may be cost prohibitive in many cases.
A wide variety of methyltransferase assays have been described in the literature ( (39). The assay utilizing a nucleosome substrate was used to screen 1,040 compounds from the Prestwick Chemical Library at a single concentration of 25 µM. While the reported assays show robust performance in 384-well format, the use of radiolabeled reagents for HTS is a challenge for many laboratories due to safety regulations and disposal costs.
Herein we report the implementation and validation of two optimized homogenous NSD2 activity assays in the highly miniaturized 1,536-well format for the identification of small molecule inhibitors from chemical libraries. The assays were utilized to screen the full-length wild-type NSD2 enzyme against a nucleosome substrate in qHTS format with three concentrations of test compounds. The use of qHTS reduces both false positive and false negative hits common to single-point HTS and facilitates selection of actives (43). For the primary screen we used the recently reported Methyltransferase-Glo assay reagent with a sensitive bioluminescent readout (44). A similar approach has been applied for the discovery of NSD1 inhibitors by HTS (67). After screening eight libraries, including numerous pharmacologically active collections, containing over 16,000 compounds, many hits were identified including chaetocin, which is known to inhibit NSD2 (39). By incorporating orthogonal and counter screens hits were prioritized for subsequent follow-up studies.
Among the confirmed active inhibitors, DA3003-1, PF-03882845, chaetocin, TC LPA5 4, and ABT-199 were selected for further characterization. In vitro potencies were determined by the HotSpot assay, which is a direct readout of the NSD2 reaction product (38). The HotSpot assay is very similar in format to the traditional gold standard radioisotope detection used in conjunction with gel electrophoresis or mass spectroscopy. The five compounds inhibited wildtype NSD2 as well as the E1099K and T1150A mutant enzymes.
DA3003-1 is a cell-permeable Cdc25 phosphatase inhibitor that potently and irreversibly inhibits all Cdc25 isoforms, including Cdc25A (IC 50 = 29 nM), Cdc25B2 (IC 50 = 95 nM), and Cdc25C (IC 50 = 89 nM) (68). It is known that DA3003-1 is capable of redox cycling (48,54), which was verified here by the Amplex Red counter screen. In addition to potent inhibition of NSD2 activity, our data demonstrates that DA3003-1 bound the SET domain with a strong affinity (K d = 370 nM, Table 3) which is comparable to its potency (IC 50 = 170 nM, Table 2). Together this suggests that DA300-1 inhibits NSD2 through a direct interaction with the catalytic SET domain, although it is most likely nonspecific. Notably,  The fungal mycotoxin chaetocin is known to inhibit NSD2 (39), so identifying it as a hit further validated our screening approach. Of the 38 methyltransferases profiled, chaetocin inhibited 12 with submicromolar potencies. Notably the methyltransferase profiling indicated chaetocin potencies of 740 nM against SUV39H1 and 570 nM against SUV39H2, which is consistent with a previously reported value of 600 nM (71). Chaetocin was initially reported to be a specific inhibitor of the histone lysine methyltransferase SU(VAR)3-9 both in vitro and in vivo (71). However, the two disulfide bonds of chaetocin can complicate bioassay interpretation because of the potential for redox activity and covalent modification of proteins (36). Indeed reports have indicated that the activity against histone lysine methyltransferases is due to chemical modification of the enzyme by the disulfide groups (72,73). Superstoichiometric binding of chaetocin to the NSD2 SET domain was observed by surface plasmon resonance, which might be due to the formation of direct compound-thiol adducts. The affinity of chaetocin to the NSD2 SET domain was strong (K d = 20 nM) with a biochemical potency about 7-fold weaker.
Chaetocin was cytotoxic to U-2 OS cells at all concentrations tested.
ABT-199, also known as GDC-0199 or venetoclax, binds BCL-2 with a subnanomolar affinity (K i < 0.010 nM) and is approved by the FDA for the treatment of CLL (74,75). The biochemical potency of ABT-199 against wild-type NSD2 (IC 50 = 1.7 µM) was similar against the NSD2 mutants, NSD1 and NSD3 (Table 4). The compound was most potent against the MLL1 and MLL4 complexes. The affinity of ABT-199 to the NSD2 SET domain (K d = 8.3 µM) is nearly 5fold weaker than the biochemical potency. At a concentration of 50 µM, ABT-199 was cytotoxic, consistent with its use in oncology, but any influences on cellular H3K36me2 levels were negligible.
DZNep is a carbocyclic analog of adenosine and a derivative of the antibiotic neplanocin-A. It was initially reported as a competitive inhibitor of S-adenosyl homocysteine hydrolase at picomolar concentrations (76). DZNep has since been reported as a global histone methylation inhibitor when used at substantially higher concentrations (36,77,78). Although DZNep was identified as a hit from the primary screen, similar activities were observed with the MTase-Glo primary and counter assays and no inhibition of NSD2 activity was observed with the orthogonal assay. The potency of DZNep in reducing H3K36me2 in U-2 OS cells (IC 50 = 390 nM; Fig. 6) is similar to its potency in reducing H3K27me3 in SU-DHL-6 cells (IC 50 = 160 nM) when assessed by the same method of western analysis (79). DZNep has been also shown to reduce H3K36me2 in SW480 cells at a concentration of 5 µM (80).
The purpose of this study was to establish a HTS discovery pipeline for NSD2, and to evaluate the workflow for identifying high-quality tool inhibitors of NSD2. The majority of the molecules screened were from pharmacologically active libraries that served to validate the primary and secondary assays. The identification of known methyltransferase inhibitors, including chaetocin and DZNep, further validated the workflow. During the course of this pilot, many known interference compounds were identified by the secondary assays, thereby demonstrating how such bad actors behave among the various assays. Five actives selected from the primary screen were shown to bind the catalytic SET domain and inhibit NSD2 activity in vitro. Although these studies confirm inhibition of NSD2, they do not rule out inhibition by intractable mechanisms of action, such as non-specific reactivity, redox, or aggregation. Two of the five compounds reduced H3K36me2 in U-2 OS cells, but the mechanisms are likely to be complicated and involving multiple targets. These studies provide a basis for the future discovery and development of novel selective NSD2 inhibitors by establishing a robust workflow for identifying and triaging hits from high-throughput screens. Upon completion, methyltransferase conversion of SAM to SAH was then detected using a twostep detection system where: 1 µL of MTase-Glo Reagent was added to each well to convert SAH to ADP for 30 min at room temperature. Finally, 5 µL of MTase-Glo Detection Solution was added to each well and allowed to incubate for 30 min at room temperature to convert ADP to ATP, which was then measured by luminescence detection using a ViewLux uHTS Microplate Imager (PerkinElmer) and compared to control samples to determine relative activity.

Methyltransferase-Glo Counterassay
MTase-Glo counterassay was performed with identical procedures as with the MTase-Glo primary assay but without NSD2 enzyme or nucleosomes. Instead 200 nM SAH was added to mimic the reaction with 20% substrate conversion.

EPIgeneous HTRF Methyltransferase Assay
The Plates were then incubated for 30 min at room temperature prior to reaction initiation with 1 µL of 4 µM [1 µM final] S-adenosyl-L-methionine (SAM) in reaction buffer and incubated at room temperature for 15 min. After the incubation period, 0.8 µL of EPIgeneous Detection Buffer One were added to each well, followed by a 10 min incubation at room temperature. Next, anti-SAH-Lumi4-Tb solution was prepared according to the manufacture's instructions and 1.6 µL of the solution were added to each well. Finally, the SAH-d2 conjugate was prepared as a 32-fold dilution according to the manufacture's instructions and 1.6 µL of the solution were added to each well. The assay plate was allowed to incubate for 1 hour at room temperature before detection of the HTRF signal using an Envision plate reader (PerkinElmer).

Amplex Red (10-Acetyl-3,7-dihydroxyphenoxazine) Assay
The assay was adapted from a previously described protocol to assess redox cycling of compounds in the presence of reducing agents (48

HotSpot Methyltransferase Assay
The HotSpot radioisotope-based methyltransferase assays were performed as described previously (83,84) with the following modifications. Standard substrate concentrations were 5 µM peptide or protein substrate, or 0.05 mg/ml for nucleosomes and core histones, and 1 µM SAM, unless otherwise mentioned. For control compound IC 50 determinations, the test compounds were diluted in DMSO, and then added to the enzyme/substrate mixtures in nanoliter aliquots by using an acoustic technology (Echo550; Labcyte) with 20 min pre-incubation. The reaction was initiated by the addition of [ 3 H]-SAM (tritiated SAM, PerkinElmer), and incubated at 30 °C for 1 hour. The reaction was detected by a filter-binding method. Data analysis was performed using GraphPad Prism software.

Surface Plasmon Resonance
The Surface Plasmon Resonance measurement was performed using Biacore 8K (GE Healthcare) at 25 °C. Human recombinant NSD2-SET domain was immobilized to a Serial-S CM5 Sensorchip (GE Healthcare) using the classic amine-coupling method in the immobilization buffer containing 10 mM HEPES, 150 mM NaCl, 0.5 mM TCEP and 0.05% v/v Surfactant P20. Single cycle kinetic measurement were performed in the running buffer containing 50 mM Tris pH 8.8, 50 mM NaCl, 0.5 mM TCEP, 5 mM MgCl 2 , 0.05% Surfactant P20 and 2% DMSO. Compounds were diluted in a 3-fold manner in the running buffer and the DMSO concentration was carefully matched to 2%. Compound solutions were then injected over the prepared sensorchip at a flow rate of 80 µL/min for 80 seconds and allowed for dissociation period of 200-600 seconds. Data analysis was performed at Biacore 8K Evaluation software using the 1:1 kinetic binding model.

Western Blot Assay
Test compounds were dissolved with DMSO to a 10 mM stock. U-2 OS cells were seeded in 12well plates at a density of 0.5 x 10 6 /well in complete culture medium and placed into the incubator at 37 °C, 5% CO 2 . After overnight incubation, the cells were treated with test compounds (10-dose with 3 fold dilution, 0.0025 -50 µM), or with reference compound DZnep (10 µM single dose) and allowed to incubate for an additional 96 hours. Following the incubation with compound, culture media was removed and the cells were washed once with ice cold PBS. The cells were lysed with 1x SDS sample buffer (62.5 mM Tris-HCl pH 6.8, 2% SDS w/v, 10% Glycerol, 0.01% w/v bromophenol blue, 50 mM DTT) and the lysates were sonicated 3 times in 3 second increments at 15 amperes. Cell lysate samples (14 µL) were subjected to SDS-PAGE and transferred onto nitrocellulose membranes by the iBlot dry blotting system. The membranes were blocked with 2% non-fat milk blocking buffer for 1 hour and then probed with anti-histone H3 (Dimethyl Lys36) primary antibody overnight. Anti-rabbit IgG IRDye 680RD secondary antibody was used to detect the primary antibody. Then the blots were washed for 3 times with 1x TBS buffer plus 0.01% Tween 20 and re-probed with anti-histone H3 primary antibody and anti-mouse IgG IRDye 800CW secondary antibody. The membranes were scanned with a LI-COR Odyssey Fc Imaging System. The specific bands of interest were quantified by LI-COR Image Studio Lite software.

U-2 OS Cell Growth Assay
U-2 OS (ATCC HTB-96) cells were obtained directly from ATCC and cultured according to the recommended culturing conditions. At cell passage 2, 1,750 cells were plated in 40 µl volumes (McCoy's 5A + 10% FBS + 0.5x pen/strep) in a 384-well cell carrier plate (PerkinElmer) and incubated overnight. At 16 hours, test compounds were delivered in 195 nL aliquots by pin transfer. The plates were sealed with Breathe-Easy sealing membrane (Sigma) and images were captured every 4 hours up to 96 hours with an IncuCyte ZOOM System (Essen BioScience).

Data Analysis
Data normalization and curve fitting were performed using in-house informatics tools. Briefly, raw plate reads for each titration point were first normalized relative to the DMSO-only wells (100% activity) and no enzyme control wells (0% activity), and then corrected by applying a plate-wise block pattern correction algorithm to remove any plate edge effects and systematic background noise. Active compounds from the primary HTS were defined as having a maximum response ≥ 50%. To determine compound activities from the 11-point qHTS, the concentrationresponse data for each sample was plotted and modeled by a four parameter logistic fit yielding IC 50 and efficacy (maximal response) values as previously described (43). The activities were designated as class 1-4 according to the type of concentration-response curve observed. Active compounds were defined as having concentration-response curves in the classes of 1-3. The promiscuity score for each compound was defined as (number of assays that the compound is active)/(total number of assays that the compound was tested in). A compound with promiscuity score higher than 0.2 was considered as a "frequent hitter" to be eliminated from the follow-up studies.