An inhibitor of KdM5 demethylases reduces survival of drug-tolerant cancer cells

Maia Vinogradova1,3, Victor s Gehling2,3, amy Gustafson1, shilpi arora2, Charles a tindell1, Catherine Wilson1, Kaylyn e Williamson2, Gulfem d Guler1, pranoti Gangurde2, Wanda Manieri2, Jennifer Busby2, e Megan Flynn1, Fei Lan2, Hyo-jin Kim1, shobu Odate2, andrea G Cochran1, Yichin Liu1, Matthew Wongchenko1, Yibin Yang1, tommy K Cheung1, tobias M Maile1, ted Lau1, Michael Costa1, Ganapati V Hegde1, erica Jackson1, robert pitti1, david arnott1, Christopher Bailey2, steve Bellon2, richard t Cummings2, Brian K albrecht2, Jean-Christophe Harmange2, James r Kiefer1*, patrick trojer2* & Marie Classon1*

The KDM5 family of histone demethylases catalyzes the demethylation of histone H3 on lysine 4 (H3K4) and is required for the survival of drug-tolerant persister cancer cells (DTPs). Here we report the discovery and characterization of the specific KDM5 inhibitor CPI-455. The crystal structure of KDM5A revealed the mechanism of inhibition of CPI-455 as well as the topological arrangements of protein domains that influence substrate binding. CPI-455 mediated KDM5 inhibition, elevated global levels of H3K4 trimethylation (H3K4me3) and decreased the number of DTPs in multiple cancer cell line models treated with stan-dard chemotherapy or targeted agents. These findings show that pretreatment of cancer cells with a KDM5-specific inhibitor results in the ablation of a subpopulation of cancer cells that can serve as the founders for therapeutic relapse.

© 2016 Nature America, Inc.

Dynamic changes in histone lysine methylation patterns mod-ulate chromatin structure and contribute to the regulation of many cellular functions, including gene expression. Notably, deregulation of histone lysine methylation has been shown to pro-mote malignant transformation and tumor progression1,2. Therefore, the enzymes that catalyze the addition and the removal of methyl groups from histone lysine residues—lysine methyltransferases and lysine demethylases (KDMs), respectively—have emerged as prom-ising oncology drug targets3–6. H3K4 is methylated by multi-protein complexes harboring SET-domain-containing histone lysine meth-yltransferases7. H3K4 is demethylated by two KDM families: the flavin-dependent monoamine oxidases LSD1 (KDM1A) and LSD2 (KDM1B), and the 2-oxoglutarate (2-OG)-dependent KDM5 hydroxylases8. The KDM5 (JARID1) demethylase family consists of four members, KDM5A–D, that all contain a catalytic Jumonji C (JmjC) domain and a number of DNA- or protein-binding domains separated by extended regions of currently unknown structure9,10. The KDM5A-encoding (KDM5A, RBP2) and KDM5B-encoding (KDM5B, also known as PLU-1) genes are amplified and/or overex-pressed in a number of human cancers, and both proteins have been described as coregulators of established oncogenes11–13. We previ-ously discovered a requirement for KDM5A in mediating cancer cell drug tolerance in non-small-cell lung cancer (NSCLC) cells14. Similarly, KDM5B was identified as a mediator of resistance to cytotoxic agents and BRAF inhibitors in melanoma15. Collectively, these findings suggest that members of the KDM5 protein family
are promising targets for drug discovery.

Crystal structures of the catalytic domains of multiple JmjC histone demethylases9,16,17 and the more distantly related protein hydroxylases18,19 reveal a funnel-shaped active site containing a compact polar binding site for the charged cosubstrate 2-OG. A catalytic iron ion and the polar amino acids, conserved across the

enzyme family, coordinate 2-OG binding, complicating the design of selective demethylase inhibitors. Nonselective metal-chelating inhibitors of these enzymes have been generated, some of which lack a clear mechanism of inhibition20–22. Other molecules that inhibit KDMs in a 2-OG-competitive manner lack selectivity23–25, complicating the interpretation of biological studies that make use of these compounds.

Here we report the discovery of a selective inhibitor, CPI-455 (1), that modulates the activity of the KDM5 family of histone demethy-lases in biochemical and cellular contexts. Furthermore, we solved the crystal structure of the intact amino-terminal half of the KDM5A enzyme including its JmjN, ARID, PHD1, catalytic JmjC and α-helical domains with and without CPI-455. Using the infor-mation from the crystal structures, we determined the mechanism of action of CPI-455 as well as its KDM5 specificity. We also show that CPI-455 specifically altered H3K4 methylation in several cell contexts. In in vitro experiments, we found that the demethyl-ase activity was required to establish drug tolerance, as treatment with CPI-455 and antitumor drugs significantly reduced the num-ber of DTPs in multiple models. Given that DTPs represent an obstacle to durable treatment responses, the data presented here would seem to describe a structure-based mechanism with possible clinical relevance.

Identification and characterization of KDM5 inhibitors

To identify inhibitors of histone demethylases, we screened a commercial compound library against the KDM4C catalytic domain (Supplementary Results, Supplementary Table 1). High-throughput screening of a 102,400-compound library against the KDM4C Jmj domain was carried out with an H3K9me3 peptide sub-strate using high-throughput mass spectrometry. Compounds were

1Genentech inc., South San Francisco, california, uSA. 2constellation pharmaceuticals, cambridge, Massachusetts, uSA. 3these authors contributed equally to this work. *e-mail: [email protected], [email protected] or [email protected]

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a b 1× OG c d
100 IC50 (µM) 6 CPI-455
O inhibition 80 20× OG 0.001 0.01 0.1 1 10 >25 µM change CPI-4203

N 1 7 6 60 KDM5A (1× 2-OG) n = 10 4
n = 6
2 Percent 40 n = 8 CPI-455 Fold 2
3 5 KDM5A (20× 2-OG)
N 4 n = 6
20 n = 6 CPI-4203
0 n = 4
N CPI-455 (1) –2 0 2 KDM5C n = 4 –2 –1 0 1 2
–4 n = 4 log [compound] (µM)
log [CPI-455] (µM) KDM2B n = 2
n = 2
N KDM3B n = 2 6 CPI-455
N 100 1× OG n = 2
n = 5 Fold change CPI-4203
Percent inhibition 80 20× OG KDM4C
N n = 7 4
60 n = 2
n = 2
N 40 KDM7B n = 2 2
CPI-4203 (4) 20 n = 4
0.001 0.01 0.1 1 10 >25 µM 0

–2 0 2 IC50 (µM) –2 –1 0 1 2
log [CPI-4203] (µM) log [compound] (µM)

100 100
Histone peptide heavy Histone peptide heavy
90 90
80 80
methyl (%) 70 methyl (%) 70
50 50
reserved. CPI-455 CPI-455
40 40
CPI-4203 CPI-4203
30 30
20 20
10 10
0 0 20
0 20 40 60 80 100 0 40 60 80 100
Time (h) Time (h)
America, Inc. All rights 100 100
Histone peptide heavy Histone peptide heavy
90 90
80 80
methyl (%) 70 DMSO methyl (%) 70 DMSO
60 60
CPI-455 CPI-455
50 50
CPI-4203 CPI-4203
40 40
30 30
20 20
10 10
0 0
0 20 40 60 80 100 0 20 40 60 80 100
Time (h) Time (h)

6 Nature Figure 1 | Identification and characterization of a potent and selective KDM5 inhibitor with reversible activity in cells. (a) chemical structures of KdM5
inhibitor cpi-455 (1, top) and the less potent control cpi-4203 (4, bottom). (b) biochemical potency of KdM5 inhibitors cpi-455, with an ic50 value of

10 ± 1 nM (top), and cpi-4203, with an ic50 of 250 ± 60 nM (bottom). Graphed data are representative single-titration curves derived from duplicate data
points, and reported ic50 values are the mean of multiple experiments ± s.e.m. (c) the selectivity of cpi-455 and cpi-4203 across a panel of recombinant
201 lysine demethylases. All assays contained 2-oG at the Km for the respective enzyme and were performed with linear reaction conditions and time-resolved
fluorescence resonance energy transfer detection (unless otherwise noted). data represent the mean ic50 values of multiple (n) replicate experiments +
s.e.m. (d) determination of the cellular potency of cpi-455 and cpi-4203 in Hela cells incubated with compounds for 4 d as measured by MSd eliSA

using antibodies specific for H3K4me3 (top) and total H3 (bottom). data are represented as the mean values from three independent experiments run
in triplicate ± s.d. (e) KdM5 inhibition affects H3K4me3 turnover. treatment with cpi-455 (20 μM) led to delayed incorporation of heavy H3K4me3
(top left) as compared to treatment with dMSo or cpi-4203 (20 μM). the turnover of H3K4me2 (top right) was marginally affected, and turnover of
H3K4me1 (bottom left) was not affected under any treatment. the graph of total histone H3 turnover (bottom right) illustrates that observed effects
on H3K4me3 turnover were not due to corresponding changes in histone H3 turnover. Representative examples are shown.

plated as orthogonal mixtures of eight compounds per well (10 μM each), with each compound represented twice. Using an initial cutoff of 35% inhibition afforded 221 primary hits. We used the dose response of the primary hits to confirm activity, and we iden-tified seven chemically tractable clusters. From these clusters, we selected 17 compounds for validation via X-ray crystallography in KDM4C. This triage process resulted in the identification of com-pound 2 (Supplementary Note, Supplementary Fig. 1a), the series precursor, as a micromolar KDM4C inhibitor with a well-defined binding mode. Screening this compound against a diverse panel of KDMs showed 2 to be a sub-micromolar inhibitor of KDM5A with approximately tenfold selectivity for KDM5A over KDM4C. Compound 2 was modified into compound 3 (Supplementary Note, Supplementary Fig. 1a), in which the 2-methyl group was replaced with a hydrogen atom. This change resulted in increased activity against KDM5A, while also increasing the selectivity of compound 3 for KDM5A over KDM4C as compared to that of compound 2. Further refinement of this chemotype resulted in the

identification of CPI-455 (1), which had improved potency against KDM5A while demonstrating ~200-fold selectivity for KDM5A over KDM4C. A detailed description of the optimization of these selective KDM5A inhibitors will be included in a later publication.

CPI-455 (Fig. 1a and Online Methods) potently inhibited full-length KDM5A in enzymatic assays with a half-maximal inhibi-tory concentration (IC50) of 10 ± 1 nM (Fig. 1b,c). A structurally related but ~25-fold less potent inhibitor of KDM5A (CPI-4203, 4; Fig. 1a–c and Online Methods) was chosen as a negative control for cellular assays. Biochemical assays using higher concentrations of the cosubstrate 2-OG showed weakened potency of CPI-455 and CPI-4203 (Fig. 1b,c), suggesting that these compounds, at least in part, are competitive with 2-OG.

To assess their selectivity, we assayed these inhibitors against a panel of recombinant, full-length KDM proteins, including KDM5A, -5B and -5C and one member from each of the five other KDM subfamilies (Supplementary Fig. 1b–d, Supplementary Tables 2 and 3). CPI-455 inhibited KDM5A, KDM5B and KDM5C to similar

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a 1.0 Unmodified

control K4 me1
0.8 K4 me2
K4 me3
DMSO 0.6
log PC9 M14 SKBR3
25 6.25 12.5 25 25 6.25 12.5 25 25 6.25 12.5 (M)

CPI-4203 CPI-455 CPI-4203 CPI-455 CPI-4203 CPI-455
b H3K4me1
H3K36me3 1.00 H3K4me2 CPI-4203, 25M
CPI-455, 6M
0.60 PC9
H3K36me2 0.40 H3K4me3 CPI-455, 12.5M

0.20 CPI-455, 25M
–0.20 CPI-4203, 25M
H3K36me1 –0.40 H3K9me1

–0.60 CPI-455, 6M M14
CPI-455, 12.5M

H3K27me3 H3K9me2 CPI-455, 25M

CPI-4203, 25M
reserved. H3K27me2 H3K9me3 CPI-455, 6M SKBR3
H3K27me1 H3K18me1 CPI-455, 12.5M
Figure 2 | CPI-455 selectively affects H3K4 methylation in several cell
models. (a) dose-dependent increase in H3K4me2/3 in pc9, M14 and
rights SKbR3 cells after 5 d of treatment with cpi-4203 and cpi-455 at the
indicated concentrations (details in Supplementary Table 4). the effect of
cpi-455 was replicated at 12.5 and 25 μM in pc9 cells, yielding H3K4me3
log2 increases of 0.49 ± 0.04 and 0.72 ± 0.07 (±s.e.m.; n = 3). (b) potency and
Inc. specificity analysis of cpi-455 in three cell line models. Quantitative changes
in histone H3 methylation after treatment with cpi-455 versus dMSo,
America, measured by mass spectrometry and expressed as log2 ratios. points along the
plot labeled “0” represent no change in the indicated mark; those inward from
0 represent quantitative decreases, and those extending outward represent
quantitative increases (details in Supplementary Table 4). Fold changes
cluster around 0 on the log2 scale; changes of 0.2 units (i.e., ±15%) have been
previously established as a threshold for significance in this analysis.
extents but showed substantially weaker potency toward KDM4C
and KDM7B (~200- and 770-fold, respectively) and no measurable
© inhibition of KDM2B, KDM3B or KDM6A (Fig. 1c). Given that

mammalian KDM5 enzymes are known to catalyze the removal of methyl groups from di- and trimethylated H3K4 (refs. 26–29), we first monitored changes in global H3K4me3 levels after compound treatment as a measure of KDM5 inhibition in cells. As expected, CPI-455-mediated KDM5 inhibition resulted in a dose-dependent increase in global H3K4me3 in HeLa cells (Fig. 1d). This compound induced a time-dependent increase in H3K4me3 that was detected only after 2 or more days of treatment (Supplementary Fig. 1f). The less potent control compound CPI-4203 did not affect H3K4me3 levels at the same concentrations (Fig. 1d). Removal of CPI-455 led to a rapid reversal of H3K4me3 increases in HeLa cells as measured by Meso Scale Discovery (MSD) ELISA (Supplementary Fig. 1e). To investigate the effect of CPI-455 on H3K4me3 formation kinet-ics, we used an approach involving methionine isotope labeling and liquid chromatography–tandem mass spectrometry (LC-MS/MS) whereby the appearance of isotopically labeled (‘heavy’) H3K4 methyl groups is measured over time30,31. CPI-455, but not CPI-4203, delayed the appearance of heavy H3K4me3 (Fig. 1e), which was consistent with a role for KDM5 enzymes in H3K4me3 turn-over. The turnover of H3K4me2 was only marginally affected (Fig. 1e). These experimental data suggest that KDM5 inhibition maintains higher levels of ‘light’ H3K4me3, which are converted into heavy methyl groups with a slower kinetics compared to DMSO-treated and CPI-4203-treated cells. As expected, neither CPI-455 nor CPI-4203 affected the turnover of H3K4 monomethylation (Fig. 1e); KDM5 enzymes are known to efficiently demethylate tri-and dimethylated but not monomethylated H3K4 (refs. 23–25).

CPI-455 specifically alters H3K4 methylation in cells
To analyze the specificity of CPI-455 in cells, we performed quan-titative mass spectrometry to determine the effects of CPI-455 and CPI-4203 on the abundance of H3K4me3 and on other histone post-translational modifications (PTMs). We measured relative fold changes in these PTMs after treatment with CPI-4203 or CPI-455 as compared to DMSO treatment (control) in melanoma (M14), breast cancer (SKBR3) and NSCLC (PC9) cell lines after 5 d of treatment. CPI-455 increased H3K4me3 and H3K4me2 in a dose-dependent manner in all cell lines tested (Fig. 2a and Supplementary Table 4). In contrast, the less potent compound, CPI-4203, did not affect these PTMs in the same the cell lines. The low baseline abundance of H3K4me2 and H3K4me3 enabled measurements of changes in

a b c
Fingers Thumb

H1 H1
H2 H2
PHD1 6 1 6 1
2 4 2 4

N 3 3

Palm C

JmjN ARID PHD1 JmjC Zn finger, PHD2 PHD3

18–60 85–176 295–341 359–588 589–787 1,163–1,216 1,614–1,659

Figure 3 | Crystal structure of KDM5A. (a) Ribbon diagram of the structure of KdM5A, colored by functional domain (see key at bottom). pHd1 was disordered in the structure, and it is schematized here by a gray oval indicating its likely location. in the schematic of domains of full-length KdM5A (bottom), a yellow oval indicates the c-terminal boundary of crystallography constructs. (b) Superposition of ARid:dnA complex from the dead ringer ARid domain (cyan, blue dnA) onto the ARid domain of KdM5A (gray). binding of dnA in this orientation would result in collision of the nucleic acid with the zinc finger and α-helical domains of KdM5A (right edge of image) within 3 bp after it lost contact with the ARid domain. the relative positions of the ARid (fingers) and the zinc finger and α-helical domains (thumb) observed in the crystal structure appear inconsistent with binding to linear or histone-wrapped circular dnA of a nucleosome particle. (c) divergent eye stereo diagram of the structural differences in the zinc-binding modules of KdM5A (color-coded as in a) and KdM6A (blue). Zinc ions are displayed as spheres.

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these marks despite the small relative decreases in the respective unmodified version of the peptide. We used antibodies to verify the dose-dependent increase in H3K4 methylation in multiple cell lines treated with CPI-455 (Supplementary Fig. 1g). The specificity of CPI-455 for the KDM5 family in biochemical assays ( Fig. 1c) was also observed in several cell lines treated with CPI-455 as indicated by the lack of change in other quantified histone PTMs (Fig. 2b and Supplementary Table 4). Together, these data show that CPI-455 is a pan-KDM5 inhibitor that is selective over other KDMs in cells and is a suitable chemical probe for further studies regarding the biological role of this enzyme family.

Structure and domain organization of KDM5A
To determine the mechanism of action of CPI-455, we crystallized a multi-domain KDM5A protein. Histone demethylase enzymes contain multiple domains that function in the recognition of bind-ing partners or substrates. However, the structures of these regions and of the catalytic Jmj domains have generally been determined in isolation. Only recently have larger segments of these complex enzymes been crystallized, shedding light on the interplay between recognition and catalytic modules. In contrast to other KDMs for which crystal structures have been reported, the KDM5 enzymes have two domains inserted between the JmjN and catalytic JmjC domains9,16. To investigate how these KDM5-specific features affect the enzyme topology and the mechanism of inhibition of CPI-455, we determined the crystal structure of KDM5A. We evalu-ated multiple crystallography constructs (Online Methods and Supplementary Table 5), and we crystallized a protein fragment encompassing residues 12–797 (KDM5A12–797) and determined its structure via the molecular replacement–single-wavelength anoma-lous dispersion (MR-SAD) method (Supplementary Tables 6–8). The 3.14-Å-resolution KDM5A crystal structure showed that the domain arrangement of this enzyme most closely resembles that of KDM6 (refs. 23,24), despite the fact that the catalytic domain shares the greatest sequence identity with the KDM4 family (33%). The topology of the KDM5A structure resembles that of a right hand, with the active site centrally located in a small pocket in the ‘palm’-region JmjC domain (Fig. 3a). This ‘palm’ region is flanked by a ‘fingers’ region composed of the ARID domain. The PHD1 domain, which is disordered in the structure, may also be part of this region. A long α-helical domain, similar to that seen in structures of KDM6 (refs. 23,24) and interrupted at its apex by a zinc-binding module, forms the ‘thumb’ region (Fig. 3a). The fold of the catalytic JmjC domain is highly conserved with that of KDM6A23,24 (PDB ID 3AVS; r.m.s. deviation = 0.46 Å over 107 Cα) and other Jmj demethylases, despite the fact that this region retains only 16% sequence identity with KDM6A.

In KDM5A, unlike what is observed in KDM4 and KDM6, the ‘left’ approach to the active site (as oriented in Fig. 3a) may be blocked by the inserted ARID and PHD1 domains, potentially affecting substrate binding. The ARID domain adopts the canonical fold but differs slightly in its loop conformations compared to the NMR structure of the isolated KDM5A ARID domain (PDB code 2JXJ32; r.m.s. deviation = 1.6 Å). The ARID domain binds double-stranded DNA and may be involved in anchoring KDM5A onto linear or nucleosome-wrapped nucleic acid. We superimposed the known structure of the ARID domain–DNA complex33 (PDB code 1KQQ; r.m.s. deviation = 1.1 Å) from the protein encoded by the Drosophila dead ringer gene ( dri) onto the ARID domain of KDM5A (Fig. 3b) and observed that the nucleic acid would project across the KDM5A structure from left to right (as oriented in Fig. 3a). However, in this orientation, the bound DNA would collide with the zinc finger domain of the thumb region 3–4 bp beyond the con-tact region with the ARID domain, suggesting a requirement for further separation of the fingers and thumb to engage nucleic acid. Docking of nucleosome cores onto the superimposed DNA revealed


N575 A411
H2O Y409

E485 Ni S478 R73





N493 Y472 A411


W503 Y409 R73





Figure 4 | Inhibitor binding at the KDM5A active site. (a) environment of cpi-455 at the KdM5A active site, color-coded as in Figure 3a. Hydrogen bonds and ion pairs are indicated by dashed lines. (b) divergent eye stereo view comparing the active sites of KdM5A:cpi-455 and KdM6b:GSK

J-1 (ref. 24) (pdb id 4ASK; dark gray) complexes. Metal ions are shown as spheres, and solvent molecules have been omitted for clarity. Amino acid differences between KdM5A, KdM4A and KdM6b are listed in Supplementary Table 9.

that neither linear nucleic acid nor circular, nucleosome-wrapped DNA would be accommodated by KDM5A without rearrangement of the relative positions of the finger and thumb regions.

The crystal structure of KDM6A23,24 revealed a single zinc-binding module of novel fold inserted within an α-helical region following the JmjC domain. The α-helical domain (Fig. 3a) of KDM5A tilts 26° farther away from the palm than that of KDM6A. The pivot point for this motion sits at residue 638, two helical turns into the rearward helix of the domain. A zinc module is also inserted within the KDM5A α-helical domain (Fig. 3a,c), but it shares limited topological similarity with that of KDM6A (r.m.s. deviation = 2.4 Å; PDB accession code 3AVS). Amino acid differ-ences between KDM5A and KDM6 generate a second zinc-binding site in KDM5A behind the β-sheet. This second zinc-binding site forms, in part, from unwinding helix H1 of KDM6A and shortening of that segment by four amino acids. The nascent pocket interacts with the metal ion via four cysteine residues, one contributed from a position that would have been on the helix of KDM6. Although the location of the other zinc site is conserved with respect to that seen in KDM6A, the ligands to the zinc consist of three cysteines and a histidine in KDM5A (Fig. 3c), as opposed to four cysteines in KDM6A. The zinc module of KDM6A coordinates the amino-terminal side (residues 17–22) of the peptide substrate for that enzyme, which demethylates histone H3 lysine (H3K27me2/3). The amino acid differences in the KDM5A zinc-binding module

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NATURE CHEMICAL bIoLogy dOI: 10.1038/nCHeMBIO.2085
create a deeper cleft at the junction of the a Parental DTP (d9) b
L-shaped peptide-binding groove observed in Relative mRNA fold change(DTP/parental) 10
KDM6, whereas a small helix formed in KDM5A 8
(replacing sheet 2 of KDM6A) would interrupt + drug 6
the path of the substrate peptide in the KDM6A 4

structure (Fig. 3c). Because KDM5A acts near
– drug 2
the amino terminus of its substrate at H3K4, the 0 m 400 0 m 400

enzyme would be unable to utilize the peptide- 0
PC9 SW480 Hs888 M14

binding mode observed for KDM6A. Therefore, it
EGFR-i Chemo BRAF-i
remains unclear whether the cleft of the KDM5A c 25 d 1.5 f
zinc module participates in protein binding of the KDM5A expression 1.0 (P = 0.06) NCI-H441

substrate peptide or in the binding to other inter- 20 Rx ecte (vem.) vehicle

acting proteins. 15 0.5
CPI-455 binds at the demethylase active site 10 0
The structure of KDM5A12–797 in complex with Relative –0.5
5 log –1.0 NCI-H441
CPI-455 revealed that the nitrile of the compound Taxol
makes a single interaction with the active-site 0 –1.5
metal ion. In addition, the compound, from the Naive neoadjuvant
NSCLC Melanoma (BrafV600E)
7-position carbonyl oxygen, forms a hydrogen e 0.3 0.2 0.2
bond with the side chain of N575 (Fig. 4a and (DTPerlotinib/parental) 0.2 (DTPAZ628/parental) (DTPlapatinib/parental)

Supplementary Fig. 2a). The central aromatic 0.1 0.1
core of CPI-455 π:π stacks with the side chains of 0 0 0
the aromatic residues Y472 and F480 and forms –0.1
an edge:face aromatic contact with W503. The Nε –0.2 –0.1 –0.1

of the side chain of K501 is positioned 3.6 Å from

–0.4 –0.2 –0.2
the partially charged (δ−) N1 of CPI-455, just out- 2 –0.5 2 2

side of hydrogen-bonding distance. The 5-phenyl log –0.6 me0 me1 me2 me3 log –0.3 me0 me1 me2 me3 log –0.3 me0 me1 me2 me3

substituent of the compound forms an edge:face
H3K4 H3K4 H3K4
aromatic contact with Y409 and points toward

solvent; the remainder of the compound is envel- Figure 5 | KDM5 activity is increased in DTP models in vitro and in vivo. (a) Microscope
oped by protein. Indeed, the 6-isopropyl moiety images showing reversible drug tolerance. nSclc line pc9 (parental; left) was treated
packs tightly in a cleft defined by the side chains with erlotinib (1 μM) for 9 d to identify dtps (right). (b) Relative fold change in amounts
of Y409 and S478 and the nearby main chain at of KDM5A mRnA in dtp cells as compared to parental cells in various models. data are
positions 410 and 473, leaving little room for addi- presented as the mean of biological triplicates ± s.e.m. (c) expression of KDM5A RnA
tional substitution. The position occupied by the in microdissected tumor cells from naive neoadjuvant nSclc biopsies. center lines indicate
inhibitor completely overlaps the binding site of means, and whiskers indicate ± s.e.m. (d) effect of vemurafenib (vem.) treatment on the
2-OG (Supplementary Fig. 2d), demonstrating a expression of KdM5A in bRiM2 trial patients (18/164) with a best objective ReciSt response
competitive mode of action, as suggested by the of progressive disease (pd) or stable disease (Sd) versus patients with a partial response (pR).
biochemical assays (Fig. 1b,c). complete responders were not biopsied in the trial. the log2 Rx effect represents a change
All amino acids within 4 Å of the inhibitor in expression after 15 d of vemurafinib treatment as compared to an untreated biopsy from
are conserved in the KDM5 family; hence, CPI- the same patient. Results shown as mean ± s.e.m. (e) A representative example of relative
455 inhibits all isoforms (Fig. 1c). The selectiv- decreases in H3K4me2/3 levels in dtps established from pc9 (left), M14 (center) and SKbR3
ity of CPI-455 for KDM5 versus KDM2, KDM3 (right) cells analyzed by mass spectrometry. data are presented as log2 ratios of dtp and
and KDM6 proteins derives from conformational parental populations. (f) immunohistochemistry for H3K4me3 in vehicle-treated (upper panel)
and sequence differences within their active sites. and carboplatin/paclitaxel-regressed (lower panel) H441 xenografts. Scale bars, 100 μm.
KDM6B is more constricted in the region flank-
ing the 5-phenyl and 6-isopropyl substituents,
where KDM6B residues K11381, F1328 and T1387 would each I73 in KDM4C. These subtle changes may explain the ~200-fold
collide with the compound (Fig. 4b and Supplementary Table 9). difference in inhibitor affinity for these two enzymes.
The KDM6B F1328 resides on a β-sheet adjacent to the site of Recently deposited crystal structures of KDM5B (Structural
insertion of the ARID and PHD1 domains in KDM5A. This Genomics Consortium; PDB ID 5A1F) appear to have used an
structural difference provides sufficient space for the equiva- internally truncated construct from which the PHD1 and ARID
lent KDM5A residue Y409 to rotate out of the pocket and permit domains were excised. These structures of KDM5B resemble those
CPI-455 binding. This conformational change may not be pos- of KDM5A in the active site and thumb region (r.m.s. deviation =
sible in KDM6B, or at least would be likely to result in a higher 0.7 Å; Supplementary Fig. 2d) but diverge at the junction where the
energy penalty for that rotation. Consistent with this structural KDM5B structures lack the ARID domain. Comparison of the struc-
difference, the dual KDM6–KDM5 inhibitor GSK-J1 binds in tures of these two isoforms does not readily explain the higher affin-
a mode partially overlapping the position of CPI-455 (Fig. 4b) ity of CPI-4203 for KDM5B, as the amino acids lining the active site
but does not enter the region occupied by the 6-isopropyl substitu- pocket are conserved in both sequence and position. Crystals were
ent, where KDM6B residue F1328 projects into the pocket. Residues not obtained with CPI-4203 and KDM5A. However, the reduced
directly contacting CPI-455 are conserved in KDM4C in the second enzyme inhibition by CPI-4203 (compared to that by CPI-455)
shell of amino acids, those surrounding the key inhibitor contact seems to arise from the concomitant removal of the methyl group
residue Y472. In particular, KDM5A A583 is replaced by S290 in in the isopropyl substituent of CPI-455, which packs well against
KDM4C. In addition, a second inhibitor contact residue, Y409, is the back of the pocket, and the likely greater twist of the phenyl
repositioned by a substitution of R73 with the branched amino acid substituent caused by the ortho-methyl substitution in CPI-4203.

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scheme. various cell line models were pretreated with cpi-455 or dMSo for 5 d and then cotreated with an anticancer agent. KdM5i, KdM5 inhibitor. (b) Representative western blots of H3K4me3 in pc9 cells (upper panels) and colo829 cells (lower panel) after 5 d of treatment with cpi-455, the cpi-4203 control compound or dMSo. (c) dose-dependent decreases in numbers of pc9 (left) and colo829 (right) dtps. dtp numbers at different doses of cpi-455 and high-dose vemurafenib (vem.) or erlotinib (erl.) were determined using live cell imaging (incucyte).
the same doses of cpi-455 had a minimal effect on the number of parental cells. data are represented as the mean of triplicate experiments + s.d. (d) percent change in numbers of parental and dtp cells after pretreatment with 25 μM cpi-455 for 5 d in various models of drug tolerance. to generate dtps, we treated pc9 cells with 1 μg ml−1 erlotnib; M14, colo205 and Hs888 cells with 2 μM AZ628; SKbR3 cells with 1 μM lapatinib; and evSA-t cells with 1 μM pi3 kinase inhibitor. this dose of cpi-455 had a minimal effect on parental cell numbers. data are represented as the mean of triplicate experiments ± s.d. (e) Representative incucyte images (cells pseudo-colored yellow) at the end-read for the highest concentration of cpi-455 as compared to dMSo in combination with targeted indicated cell models.

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article NATURE CHEMICAL bIoLogy dOI: 10.1038/nCHeMBIO.2085

In summary, the structure of KDM5A pro- a KDM5i + anti-cancer agent c Parental Parental

vided insight into the mechanism of inhibition 1.2 DTP (erl.) 1.6 DTP (vem.)

as well as the selectivity of CPI-455. 5 d 6–15 d 1.0 1.4
KDM5 inhibition results in fewer b 0 6.25 12.5 25 CPI-455 (M) Fold change 0.8 Fold change 1.2
H3K4me3 1.0
drug-tolerant cells PC9 0.6 0.8
Given the cellular potency, specificity and Histone H3 0.4 0.6


clear mode of binding of CPI-455, we used the 0.2
compound to evaluate the role of the KDM5 0 6.25 12.5 25 CPI-4203 (M) 0.2
0 0
demethylase activity in the establishment of PC9 H3K4me3 0 6.25 12.5 25 051025
drug tolerance14,15,34. We previously defined a Histone H3 CPI-455 (M) CPI-455 (M)
drug-tolerant state (DTPs; Fig. 5a) that gives e
rise to drug-tolerant expanded persister can- M M M
 M
cer cells (DTEPs). We have also reported that 10   Colo205
 10 25
DTEPs show increased expression of KDM5A, -4203 – 455 -455 -455
and that the emergence of these populations ControlDMSO CPI CPI CPI CPI
is dependent on KDM5A, as knockdown

results in a reduction in the number of emerg- Histone H3 -T
ing DTEPs in response to various agents in EVSA
an NSCLC cell line14. Here we found a gen-
eral increase in KDM5A RNA levels in sev- NSCLC Melanoma Colon Breast
eral in vitro DTP models (Fig. 5b), as well as % change in cell number after-455treatment(DMSO/CPI-455) 50 PC9 Hs888 M14 Colo205 SKBR3 EVSA-T
SKBR3 Hs888
in regressed melanoma (Fig. 5c) and NSCLC

(Fig. 5d) tumors compared to levels seen in 0

nonresponding and untreated patient tumors,
respectively. We have previously shown by
western blot analysis that H3K4me3 is reduced –50

in DTPs from the NSCLC PC9 cell line14. Here

we observed demethylation of H3K4me2/3 by

histone MS in multiple DTP models, including –100 Parental
PC9 cells (Fig. 5e). Similar decreases could also CPI DTP PC9

be seen in drug-regressed xenografts (Fig. 5f CPI-455 25M

and Supplementary Fig. 3a). Taken together,
these findings suggest that the demethylase
activity of KDM5 is required for the survival of Figure 6 | KDM5 inhibition suppresses the emergence of drug-tolerant cells. (a) treatment

drug-tolerant cells. To test this directly, we first expressed either a wild-type or a demethylase dead mutant (H483A) FLAG-tagged KDM5A protein in PC9 cells. The cells were subse-quently infected with a short hairpin construct targeting the 3′ UTR of KDM5A also express-ing GFP (Supplementary Fig. 3b). After selection of drug-tolerant cells with erlotinib (1 μM), the only cells that maintained expres-sion of the KDM5A short hairpin (GFP) were cells that expressed the exogenous wild-type KDM5A protein (Supplementary Fig. 3c), suggesting a requirement for the demethylase activity of KDM5A.

To test the importance of the KDM5 demethylase activity in DTP survival using
CPI-455, we first established an assay that could detect these rare other cell line models treated with a variety of drugs (Fig. 6d,e).
cells (Online Methods). For this purpose, we used two inhibitors The reduction in the number of DTPs in cell populations treated
that previously have been shown to diminish the outgrowth of with CPI-455 was dose-dependent in all the cell models tested
DTEPs, the HDAC inhibitor TSA and the IGF-receptor inhibitor (Supplementary Fig. 3g,h). These cell lines represent various can-
AEW-541 (ref. 14). Both of these inhibitors reduced the number cer types that are treated with different targeted or chemothera-
of DTPs (Supplementary Fig. 3d,e) without having an apprecia- peutic agents. Importantly, the decreases in DTP numbers were
ble effect on the parental population (Supplementary Fig. 3d,e). observed at concentrations of CPI-455 that did not affect the pro-
After these experiments, we treated PC9 (NSCLC) cells and colo829 liferation or survival of the parental cell population (Fig. 6d and
(melanoma) cells with CPI-455 to increase H3K4me3 before the Supplementary Fig. 3g,h). Previous studies have shown that DTPs

addition of a targeted agent (Fig. 6a,b). Pretreatment with CPI-455 originate in a subpopulation of cells within heterogeneous parental
resulted in a dose-dependent decrease in the number of DTPs in cancer cell populations that exhibit increased aldehyde dehydro-
PC9 cells treated with erlotinib and colo829 treated with vemu- genase activity35, and in the current study treatment with CPI-455
rafinib (Fig. 6c). CPI-455 did not have an appreciable effect on the in several cancer cell lines resulted in a decrease in the number of
parental population in either case (Fig. 6c). The CPI-455-induced aldehyde-dehydrogenase-expressing cells in the parental population

decrease in the number of surviving DTPs was also seen in several (Supplementary Fig. 3f). Collectively, these findings demonstrate

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NATURE CHEMICAL bIoLogy dOI: 10.1038/nCHeMBIO.2085 article

© 2016 Nature America, Inc. All rights reserved.

that the survival of drug-tolerant subpopulations of cancer cells seemingly requires the demethylase activity of the KDM5 family during otherwise lethal drug exposures.

Targeting histone lysine methylation pathways has recently emerged as a promising strategy for cancer treatment. Multiple histone lysine methyltransferase inhibitors have been described, and some of them have progressed into clinical trials2. However, so far the only reported advanced KDM inhibitors target the FAD-dependent demethylase (KDM1A) class of enzymes. Identifying selective, drug-like inhibitors of JmjC-type, 2-OG-dependent demethylases has proven difficult, and current KDM inhibitors either lack selectivity or have an unclear mechanism of action. Here we report the iden-tification of CPI-455, a selective inhibitor of KDM5A. CPI-455 acts as a pan-KDM5 inhibitor and thus has the potential to overcome functional redundancies among KDM5 family members in disease-relevant contexts. CPI-455 is 200-fold selective over the most closely related KDM4 enzymes and >500-fold selective over other JmjC-type demethylases, with the observed biochemical specificity reca-pitulated in cellular assays. The KDM5A cocrystal structure with CPI-455 demonstrated the mechanism of action of the inhibitor, the basis for its selectivity versus related enzymes, and provided guid-ance for future improvement of this chemical series. Additionally, the KDM5A crystal structure provides the most complete structure of these multidomain enzymes to date. The present structure, along with recent structures of KDM3, KDM6 and KDM7 (refs. 23,24,36), offers glimpses into the varied topological organization of these complex enzymes.

Drug resistance remains a challenge to successful cancer therapy, and it has been suggested that the KDM5 family of demethylases has a role in the emergence of drug tolerance14,15,34. The inhibitor CPI-455 possesses the target specificity required for an in vitro tool compound for exploring KDM5-dependent disease biology, includ-ing drug tolerance. Here we show that the demethylase activity of KDM5 is important for DTP survival, as CPI-455 reduced the number of surviving cells after lethal drug exposures in a number of cell culture models. Future studies with more potent inhibitors will enable in vivo experiments as well as expanded studies of potential single-agent activities of these compounds.

received 15 July 2015; accepted 6 april 2016; published online 23 May 2016

Methods and any associated references are available in the online version of the paper.

Accession codes: Coordinates and amplitudes for the crystal struc-ture have been deposited at the RCSB PDB under accession 5CEH.

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We thank the Genentech Structural Biology Expression Group and J. Wu for additional protein expression and purification, as well as other members of the KDM5 team.
We also thank J. Settleman for comments on the manuscript and B. Haley for short hairpin design. We thank Shamrock Structures, LLC, for diffraction data collection from beamline 08ID-1 at the Canadian Light Source, supported by the Natural Sciences and

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NATURE CHEMICAL bIoLogy dOI: 10.1038/nCHeMBIO.2085

Engineering Research Council of Canada, the National Research Council

Canada, the Canadian Institutes of Health Research, the Province of Saskatchewan, Western Economic Diversification Canada, and the University of Saskatchewan, and from beamline 5.0.2 of the Advanced Light Source. The Berkeley Center for Structural Biology is supported in part by the National Institutes of Health, National Institute of General Medical Sciences, and the Howard Hughes Medical Institute. The Advanced Light Source is supported by the Director, Office of Science and Office of Basic Energy Sciences, of the US Department of Energy under Contract No. DE-AC02-05CH11231.

G.D.G., H.K., T.L., M.C. and R.P. were involved in the design and execution of DTP and ALDH assays. M.W., Y.Y., E.J. and G.V.H. were involved in the in vivo analysis of tumors in patients and mice. T.K.C., T.M.M., D.A. and J.B. participated in histone MS analysis. J.R.K., P.T. and M.C. contributed equally, oversaw all experiments and wrote the manuscript.

Competing financial interests

The authors declare competing financial interests: details are available in the online version of the paper.

author contributions

M.V. and J.R.K. were involved in the crystallography efforts. V.S.G., A.G., K.E.W., P.G., W.M., E.M.F., F.L., S.O., A.G.C., Y.L., C.B., S.B., R.T.C., B.K.A. and J.-C.H. were involved in assay development, protein production, biochemical assays, and the development and execution of cell-based assays. F.L. made the KDM5A constructs. S.A., C.A.T., C.W.,

additional information

Any supplementary information, chemical compound information and source data are available in the online version of the paper. Reprints and permissions information is available online at http://www.nature.com/reprints/index.html. Correspondence and requests for materials should be addressed to J.R.K., P.T. or M.C.

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Expression and purification of full-length KDM proteins. The cDNA of human full-length KDMs (UniProt IDs: P29375 (KDM5A), Q9UGL1 (KDM5B), P41229 (KDM5C), Q8NHM5 (KDM2B), Q7LBC6 (KDM3B), Q9H3R0 (KDM4C), O15550 (KDM6A) and Q9UPP1 (KDM7B)) was cloned in-frame with the N-terminal FLAG tag in pBlueBac 4.5 vector by Gateway trans-fer technology (Thermo Fisher Scientific). Virus production was performed by Kinnakeet Biotechnology LLC (Midlothian, VA). A 2-L flask containing 1.8 × 106 cells/ml in serum-free Sf-900 SFM (Gibco) was inoculated with recombinant baculovirus (multiplicity of infection ~ 2–3) for 40–44 h. The cells were lysed using the Branson Sonic Dismambrator (Fisher Scientific) in buffer containing 30 mM Tris, pH 7.4, 0.3 M NaCl, 0.2% Triton X-100, 0.5 mM TCEP and Complete EDTA-free protease inhibitors cocktail (Roche). The cell lysate was cleared by centrifugation at 20,000g for 30 min, and the supernatant was applied to anti-FLAG M2 affinity gel (Sigma), equilibrated in lysis buffer. The protein–bead mixture was incubated for 3.5 h at 4 °C with gentle mix-ing. At the end of the binding step, the protein–bead mixture was extensively washed in lysis buffer with Econo-Pac chromatography columns (Bio-Rad). FLAG-tagged proteins were eluted from the affinity gel by peptide competition with 2.5 column volumes of elution buffer, 30 mM Tris-HCl, 0.3 M NaCl, 0.02% Triton X-100, 0.5 mM TCEP containing 0.15 mg/ml 3×FLAG peptide (Sigma). The 3×FLAG peptide was removed by dialysis, and purity was assessed by Coomassie gel and western blotting (Supplementary Fig. 1b).

KDM4C enzymatic screening assay. High-throughput MS assays were car-ried out using truncated KDM4C in 384-well V-bottom polypropylene plates (Greiner, Inc.). His-tagged recombinant KDM4C (1–350) was overexpressed and purified in-house from Escherichia coli BL21(DE3) to near-homogeneity. The demethylation reaction buffer contained 50 mM Tris-HCl, pH 7.5, 0.01% Triton X-100, 5% glycerol, 1 mM sodium ascorbate (Sigma), 5 μM 2-OG (Sigma) and 20 μM Fe2(NH4)2(SO4)2 (Sigma). Compounds were added to the plates from 10 mM DMSO stocks either as mixtures of eight compounds for screen-ing (25 nl each, 10 μM final) or as serial dilutions of single compounds for IC50 determinations. Additional DMSO was added as a backfill (final DMSO: 0.8% (vol/vol)) using a LabCyte Echo 550. In 25-μl demethylation reactions, 400 nM recombinant KDM4C and 20 μM H3K9me3 peptide (1–21 amino acids, New England Peptide) were incubated with compounds for 10 min, and then 2-OG and Fe2(NH4)2(SO4)2 were added to initiate the reaction. All of the reactions were incubated for 45 min at room temperature and then quenched with 25 μl of 1 N HCl. After termination, plates were sealed, frozen at −80 °C and shipped on dry ice for analysis using the RapidFire high-throughput MS platform (BioTrove Inc., Woburn, MA). Briefly, plates were thawed and immediately analyzed using the RapidFire system coupled to a Sciex API4000 triple-quadrupole mass spectrometer. The samples were delivered directly from the plate to a clean-up cartridge (BioTrove column A) to remove nonvolatile assay components with 0.1% formic acid in a 3-s wash cycle. The peptide substrate and demethylated product were coeluted to the mass spectrometer with 80% acetonitrile, 0.1% formic acid. Both substrate and product signals were read at their +5 charge species, and the conversion from substrate to product was assessed by [H3K9me2 Read]/[H3K9me2 Read + H3K9me3 Read].

KDM5 enzymatic assays. Time-resolved fluorescence resonance energy trans-fer (TR-FRET) assays were carried out using full-length KDM5 enzymes in 384-well black ProxiPlates (PerkinElmer Inc.). The indicated final reaction concentration of KDM5A (2 nM), KDM5B (8 nM) or KDM5C (4 nM) was combined with 2-OG (Sigma) at Km (final reaction concentration of 1 μM for KDM5A and KDM5C and of 2 μM for KDM5B, unless otherwise indi-cated (20× = 20 μM)) in assay buffer (50 mM HEPES, pH 7.0, 2 mM DTT, 0.5 mM ascorbic acid and 0.01% (vol/vol) Triton-X-100) in a total volume of

2.5 μl. To this we added 2.5 μl of compound that had previously been diluted in ten-point serial dilutions in DMSO and then diluted in assay buffer to 1% DMSO (vol/vol, final). Reaction was initiated by the addition of 5 μl of 200 nM H3K4me3 peptide substrate stock (ARTK(me3)QTARKSTGGKAPRKQLA-NovaTagPEG-biotin, New England Peptide) in assay buffer containing 200 μM (NH4)2Fe(SO4)2 hexahydrate (Sigma). Reactions were carried out for 30–45 min at room temperature and were stopped by the addition of 10 μL of detection

reagents, 100 nM SA-XL665 (CisBio Inc.) and 1 nM Eu3+-labeled anti-H3K4me1/me2 (PerkinElmer Inc.) in 50 mM Tris, pH 7.5, 2 mM EDTA, 0.01% BSA (wt/vol) and 0.01% Triton X-100. Plates were spun (20 s, 1,000 r.p.m.), incubated for 1 h at room temperature and read on an Envision reader (PerkinElmer Inc.) using manufacturer-recommended settings, filters and mirror. XLFit 5 software (IDBS) was used to generate graphs and to fit curves. We determined IC50 values by fitting the percent inhibition versus compound concentration relative to positive and negative controls using a four-parameter fit equation.

KDM selectivity panel. TR-FRET assays similar to those described above for KDM5 enzymes were carried out with full-length KDM2B, KDM3B, KDM4C, KDM6A and KDM7B. For substrates and enzyme concentrations, see Supplementary Table 2.

Cell culture. HeLa, Colo829 and U2OS cells were obtained from ATCC (Manassas, VA). Fetal bovine serum (FBS), penicillin–streptomycin (Pen– Strep) and other cell culture reagents were purchased from Life Technologies (Carlsbad, CA). HeLa and U2OS cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated FBS and 1× Pen–Strep, whereas Colo829 cells were grown in RPMI 1640 medium sup-plemented with 10% FBS and 1× Pen–Strep. All other cell lines were grown in high-glucose (4.5 g/L) RPMI media supplemented with sodium pyruvate, L-glutamine and 10% FBS (Sigma). The serum concentration in inhibitor experiments was 5%. Cell line identity was routinely monitored by SNP-based genotyping at Genentech.

DTP assays. All cell lines were treated with DMSO, CPI-4203 or CPI-455 for 5 d with two changes of medium and drug. Thereafter, the cells (PC9, Colo205, Hs888, M14, SKBR3 and EVSA-T) were plated at 2 × 105 cells in six-well plates in triplicate and treated for an additional 9–15 d, depending on the cell line model. The Incucyte HD imaging system (Essen Biosciences) was used to monitor numbers of drug-tolerant cells after cells were stained with Nuclear-ID Red stain (Enzo). In the representative images in Figure 6f, these signals are converted to yellow. Colo829 cells were treated with 20 μM vemu-rafenib (PLX4032; Selleck Chemicals, Houston, TX). We treated PC9 cells with 1 μM erlotinib, M14 and Hs888 cells with 2 μM AZ628, and SW480 cells with 5-FU (36 μM) and an irinotecan active metabolite (SN38, 6 nM). In all cases, medium and drug(s) were changed every 3 d. For the Colo829 line, cells were transduced with CellPlayer NucLight Red (Lenti, EF-1 alpha, bleo; Essen Bio, Ann Arbor, MI) to obtain a pure population of NucLight Red–expressing cells. These cells were plated (1.5 × 105 cells/ml) in 6- (3 ml), 12- (2 ml) and 24-well (1 ml) plates. After 2 d, vemurafenib (20 μM) or DMSO was added to replicate wells (for this experiment, no antibiotic was added to the medium) and the plates were incubated within the Incucyte chamber to quantify live cell num-bers every 6 h until the completion of the experiment (8 d after the addition of vemurafenib). Medium was replaced with fresh vemurafenib-containing medium every 3 d. For experiments with the HDAC inhibitor TSA (Sigma) or the IGF-receptor inhibitor AEW541 (Selleckchem), the cells were cotreated with various concentrations of TSA or AEW541 and erlotinib (1 μM) for 8 d.

Knockdown rescue experiment. PC9 cells were transfected with inducible FLAG-tagged KDM5 wild-type and H483A mutant. Thereafter, the cells were infected with a lentiviral construct containing GFP, a scrambled or a KDM5A 3′ UTR–directed short hairpin (TAAGCCTCTAACTACTATCAG) containing GFP. Expression of exogenous and endogenous KDM5A in selected pools of cells was analyzed using semiquantitative RT-PCR with the following primers: KDM5A_3′ UTR (forward), GTTAGTGTTGCTGTGCATATGT; KDM5A_3′ UTR (reverse), TTGCCAGAGTTAAAATACTAATGATG; KDM5A-FLAG (forward), GGACTACAAAGACCATGACG; KDM5A-FLAG (reverse), AGGA TTTTTCTCTCTACCACAG; hGAPDH (forward), GAGTCCCTGCCACAC TCA; hGAPDH (reverse), GGGGTCTACATGGCAACTG. The cells were treated for 30 d with erlotinib (1 μM) and analyzed for the expression of GFP as a marker of short hairpin activity. Cells that emerged in the pools that expressed wild-type KDM5A were green, whereas cell populations that formed DTEPs in the pools expressing mutant KDM5A were not, suggesting a requirement for

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KDM5A demethylase activity in the formation of DTEPs with reduced endog-enous KDM5A expression.

Aldefluor staining. Aldefluor staining was performed according the manufac-turer’s recommendations (Stem Cell Technologies). In short, media was aspi-rated and 1 ml of assay buffer including 5 μl of Aldefluor reagent was added per well in six-well dishes. After incubation for 30 min at 37 °C, buffer was aspirated and cells were washed twice with PBS. Wells were imaged using IncucyteZoom (Essen Bioscience). The data from PC9, M14 and SKBR3 cells treated with CPI-4303 or CPI-455 are shown in Supplementary Figure 3f.

Western blotting. For western blotting, we prepared nuclear extracts from cells by first separating the cytoplasmic fraction using buffer A (10 mM Tris, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 25 mM NaCl, 0.5 mM DTT, 0.2 mM phenylmeth-anesulfonyl fluoride (PMSF), and protease inhibitors (Complete mini, Roche)). We subsequently isolated the nuclear fraction using buffer B (25 mM HEPES, 420 mM NaCl, 20% glycerol, 1.5 mM MgCl2, 0.1 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF, and protease inhibitors). All antibodies used for western blot-ting are described in Supplementary Table 3.

Quantitative reverse-transcription PCR (qRT-PCR). qRT-PCR was carried out according to standard protocols. Briefly, we extracted RNA using the RNeasy kit (Qiagen) and then carried out cDNA synthesis using SuperScript III reverse transcriptase (Invitrogen, Carlsbad, CA). This cDNA was used for qPCR reac-tions using gene-specific primers and SYBR Green chemistry or Fluidigm anal-ysis. Cyclophilin B, GAPDH, actin or RPL19 was used for normalization. For analysis by Fluidigm, isolated RNA was subjected to a one-step cDNA synthesis using the iScript cDNA synthesis kit (Bio-Rad). cDNA was subsequently pre-amplified using the Taqman PreAmp Mastermix (ABI 4391128) according to the manufacturer’s protocol, except that PCR cycling conditions were modified to a 14-cycle pre-amplification step. After amplification, samples were diluted 1:4 with TE (10 mM Tris-HCl, 1 mM EDTA), and qPCR was conducted on Fluidigm 96.96 Dynamic Arrays using the BioMark HD system according to the manufacturer’s protocol. Samples were run in triplicate, and cycle threshold values were converted to relative expression values using GAPDH and RPL-19 as controls. Taqman probes used were GAPDH (Hs02758991_g1), KDM5A (Hs00231908_m1), KDM5B (Hs00981910_m1) and RPL19 (Hs02338565_gH).

Nanostring RNA analysis of patient tumors. BRIM2(NCT00949702) was a single-arm phase 2 study in which patients (132) carrying a BRAFV600E/K mutation received vemurafenib. Tissue blocks collected before and after therapy from the same patients were available for 18 patients from the study. Patient consent was obtained for exploratory research conducted on all tis-sues. mRNA was prepared from formalin-fixed, paraffin-embedded sections of tumor tissue, and gene expression was measured using nanostring. Data were normalized to the geometric mean of all 800 genes profiled. The effect on baseline expression was determined using a Cox proportional hazards model. Naive and post-treatment frozen tumor samples were obtained from BioOptions, Inc., and Indivumed, Inc. For tissues from NSCLC biopsies, sections were stained with hematoxylin and eosin, and epithelial cells identi-fied on the basis of morphology were collected by laser-capture microdissec-tion using the Cellcut Plus system (MMI). RNA was isolated using the RNEasy micro kit (Qiagen), and cDNA synthesis was performed with the Advantage RT for PCR kit (Clontech), per the manufacturer’s instructions. cDNA was subject to 19 cycles of pre-amplification using Taqman PreAmp Master Mix and a pool of 0.2× Taqman assays (Applied Biosystems). qRT-PCR was con-ducted on the BioMark HD system using 48.48 Dynamic Arrays (Fluidigm) according to the manufacturer’s protocol (as above). Data were analyzed using the Fluidigm Real Time PCR Analysis software.

In vivo immunohistochemistry study. Calu3 study. Fifteen million Calu3 cells were transplanted subcutaneously into the right flank of athymic nude mice (female, >8 weeks old, n = 12 per group). When tumors reached 100–300 mm3 in size, mice were divided into two treatment groups with equal average starting volumes. Group 1 received Paclitaxel vehicle in 5% EC (ethanol/camphor) i.v. every other day for five doses (10 d) and received PBS i.p. on days 1 and 7. Group

2 received chemotherapy in the form of Paclitaxel (20 mg/kg) i.v. every other day for five doses (10 d) and Cisplatin (5 mg/kg) i.p. on days 1 and 7. Tumors were collected at day 17 and processed for histopathological evaluation.
H441 study. Twenty million H441 cells were transplanted subcutaneously into the right flank of athymic nude mice (female, >8 weeks old, n = 12 per group). When tumors reached 100–300 mm3 in size, mice were divided into two treatment groups with equal average starting volumes. Group 1 received Paclitaxel vehicle in 5% EC i.v. every other day for five doses (10 d) and PBS i.p. on days 1 and 14. Group 2 received chemotherapy in the form of Paclitaxel (20 mg/kg) i.v. every other day for five doses (10 d) and Cisplatin (5 mg/kg) i.p. on days 1 and 14. Tumors were collected at day 21 and processed for histopathological evaluation using an immunohistochemistry-validated rab-bit monoclonal antibody from Cell Signaling Technologies (antibody details provided in Supplementary Table 3) . All animal experiments were approved by the Institutional Animal Care and Use Committee at Genentech Inc.

MSD ELISA. Cells were plated in 96-well plates and treated with various con-centrations of CPI-455 or CPI-4203 for up to 4 d. Cells were harvested at 24, 48, 72 and 96 h after compound addition. For compound wash-out studies, cells were rinsed with medium 96 h after the initial addition of compounds, and fresh medium without compounds was added. Cells were collected at various time points, and global levels of H3K4me3 and total H3 were determined using MSD ELISA. MSD ELISA was carried out as previously described37. Briefly, after removal of medium, cells were rinsed with PBS and lysed with a hypo-tonic buffer (10 mM HEPES, pH 7.9, 5 mM MgCl2, 0.25 M sucrose, Benzonase (1:10,000, Roche), 1% Triton X-100 supplemented with 1× protease inhibitor cocktail (Roche) and 1 mM PMSF) followed by addition of NaCl to a final concentration of 1 M. The lysates were diluted with Tris buffer (20 mM Tris, pH 7.5, 1 mM EDTA, 1 mM EGTA) supplemented with 1× protease inhibi-tor cocktail and 1 mM PMSF, then adjusted to a final NaCl concentration of 100 mM. MULTI-ARRAY microplates (L15XA-3, MSD; Rockville, MD) were coated with histone-capture antibody (2 μg/ml for H3K4me3 capture and 1 μg/ml for total H3 capture) and blocked for 1 h with 5% BSA solution before the addition of samples. After 3 h of incubation, the lysates were discarded and the plates were washed three times with Tris-buffered saline containing 0.02% Tween-20. H3K4me3 and H3 levels in the captured histones were detected using appropriate primary and secondary antibodies and a SECTOR Imager 2400 instrument (MSD; Rockville, MD). GraphPad Prism 6.0 was used to plot data and calculate EC50 values.

Histone-modification mass spectrometry. PC9, M14 and SKBR3 cell lines were treated for 5 d with DMSO or KDM5 inhibitors at varying concentra-tions (CPI-4203 at 25 μM; CPI-455 at 6.25 μM, 12.5 μM and 25 μM, except in SKBR3 cells, which did not receive 25 μM CPI-455 because there was some cytotoxicity at that concentration). We also collected pellets for parental and DTP populations. Cell pellets were collected (2–5 × 106 cells), and core histones were extracted and purified using a commercial kit (Histone Purification Mini Kit, Active Motif, Carlsbad, CA). Purified histones were desalted by perchlo-ric acid precipitation and re-dissolved in water to a concentration of approxi-mately 1 μg/μL, and aliquots were stored at −80 °C. Histones were prepared for mass spectrometric analysis as described previously38. Briefly, unmodified and mono-methyl lysines were capped by reaction with propionic anhydride under mild aqueous conditions and then subjected to tryptic digestion to produce arginine-terminated peptides. Peptide neo-N termini were blocked with phe-nyl isocyanate (samples) or 13C6-labeled phenyl isocyanate (histone standards). Each sample was mixed with an equal amount of the stable-isotope-labeled histone standard and desalted by C18 StageTip (Thermo Fisher Scientific, West Palm Beach, FL). Peptide mixtures were injected via autosampler for microcap-illary reverse-phase chromatographic separation (NanoAcquity UPLC; Waters Corp., Dublin, CA) in line with the electrospray-ionization source of a hybrid ion trap–orbitrap mass spectrometer (Orbitrap Fusion with ‘Easy-Spray’ source; Thermo Fisher). Chromatography was performed with a PepMap RSLC C18 column (2-μm beads, 100-Å pores, 75-μm inner diameter by 15 cm; Thermo Fisher). Peptides were eluted in a 35-min gradient from 2% vol/vol solvent B to 25% solvent B at 0.5 μL per minute, where solvent A was 2% vol/vol acetonitrile and 0.1% vol/vol formic acid in water, and solvent B was 2% water, 0.1% formic

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acid in acetonitrile. Both full-scan mass spectra (m/z 325–1,200 with resolution of 60,000 at m/z 195) and targeted product-ion spectra (parallel reaction moni-toring; m/z 100–1,800 with 15,000 resolution at m/z 195) were performed in each duty cycle. Ions targeted for tandem mass spectrometry corresponded to 61 histone H3 peptides encompassing 24 methyl and acetyl marks in their various combinations in a ‘scheduled’ manner according to retention times and with activation by higher-energy collisional dissociation at 30% relative colli-sion energy and 2.0 m/z isolation windows. Quantitative data were extracted from the mass spectra using the Skyline application39 and converted to rela-tive abundances for each peptide and histone mark as previously described38. Intersample normalization was accomplished using the stable-isotope-labeled internal standards, and fold changes in each mark were calculated as log2 ratios versus DMSO control.

Histone mass spectrometry turnover experiment. For methyl turnover experiments, HeLa cells were seeded at 1.5 × 106 cells per p100 dish in DMEM complete media. The next day, the media was replaced with DMEM without cysteine, glutamine or methionine (Life Technologies) and supplemented with L-cysteine dihydrochloride (Sigma C6727) at 65.2 mg/L, L-glutamine (Sigma G8540) at 300 mg/L, L-methionine-(methyl-13C,d3) (Sigma 299154) at 15 mg/L, 10% FBS (HyClone) and antibiotics (Invitrogen), and cells were grown in the presence of CPI-455 or CPI-4203 at 20 μM, or with 0.25% DMSO. Cells were trypsinized from the culture plates, pelleted by centrifugation at 500g for 3 min, washed with PBS, and pelleted again. Cell pellets were snap-frozen and stored at −80 °C until processed further. For all cell pellets to be analyzed by mass spectrometry, the histone isolation was performed using the EpiQuik kit (Epigentek, Farmingdale, NY) per the manufacturer’s instructions. Resultant soluble histone protein was precipitated using 20% TCA for 3 h on ice. After isolation, histones were propionylated (Sigma, St. Louis, MO) as previously described40 to block free amines on the histone proteins and then treated with trypsin (Promega, Madison, WI) for overnight digestion. Peptides were then subjected to acylation with phenylisocyanate to block the termini created from the digestion38. Next, each sample was desalted using a StageTip, with an elu-tion of 25% MeCN before analysis by Orbitrap MS. Peptides were re-suspended in 0.1% formic acid and analyzed by direct online injection into a Q Exactive mass spectrometer (Thermo Scientific). Chromatographic separation was performed over a gradient of 0–55% acetonitrile containing 0.1% formic acid over 55 min on a 100 μm × 15 cm analytical column packed with Magic C18 packing material (New Objective, Woburn, MA) with a flow rate of 300 nl/min. Peptides were sprayed using a 20-μm emitter tip using a spray voltage of 2.0 kV and a capillary temperature of 200 °C. Survey scans were acquired in the range of m/z 300–1,750 at 70,000 resolution and with an automatic gain control target of 1 × 106 with polydimethylcyclosiloxane from ambient air (m/z 445.120025) as a lock mass41. For each cycle, the ten most intense ions were fragmented using higher-energy collisional dissociation with a normalized collision energy of 27.0 at a target of 5 × 104 ions or 50 ms maximum injection time before being excluded for 30 s.

Heavy- methyl incorporation. Masses for each potential methylated form of the H3 3-8 peptide were calculated for tracking in the study. These masses were used to generate single-ion chromatogram plots in XCalibur QualBrowser (Thermo Scientific) to determine the elution of each peptide and the area under the curve (AUC) of the eluting species. For each eluting peptide, the charge state and accurate mass of the peptide were confirmed with the mass expected to be found with less than 10 ppm error. AUC information for each peptide peak was cataloged for further calculations, and the peptide identi-fication and location of modification were determined from MS/MS scans. After all peptide species had been identified in the analyses, the AUC data were used to calculate the percentage of each species that contained heavy methyl groups42. We derived the data represented in Figure 1f by determining the abundance of peptide species for which all of the respective H3K4 methyl groups were isotopically labeled (‘Histone peptide heavy methyl’).

Enzymatic peptide demethylation assay. We used a TR-FRET activity assay to monitor the proximity of anti-H3K4me2 (donor) and streptavidin (acceptor) molecules. Fluorescence intensity increases as donor and accep-tor associate with dimethylated peptide produced by demethylation of a

trimethylated substrate. The biotinylated peptide substrate (H2N-ART(KMe3) QTARKSTGGKAPRKQLA-NTPEG-biotin) and product control (H2N-ART (KMe2)QTARKSTGGKAPRKQLA-NTPEG-biotin) were custom synthesized by New England Peptide (Gardner, MA). Streptavidin-XL665 and histone H3K4me2 monoclonal antibody (Supplementary Table 3) labeled with euro-pium cryptate were from CisBio (Bedford, MA). Enzyme reactions were car-ried out with 100 nM peptide substrate, 3 μM α-ketoglutaric acid sodium salt (2-OG, Sigma), 100 μM ammonium iron (II) sulfate hexahydrate (Sigma) and up to 400 nM enzyme in reaction buffer (50 mM HEPES, pH 7.0, 0.01% Triton X-100, 0.01% BSA, 500 μM sodium-L-ascorbate) at ambient temperature for 35 min. The reactions were quenched by the addition of equal volume detection (1 nM H3K4me2-Eu(K), 50 nM SA-XL665, 50 mM Tris-HCl, pH 7.5, 0.01% Triton X-100, 233 mM KF, 2.3 mM EDTA). After 1 h at ambient temperature, the TR-FRET ratio with 320 nm excitation, 615 nm donor emission, and 665 nm acceptor emission was calculated as (665 em/615 em) * Z, where Z = 5,000. The percent substrate conversion was calculated relative to the 100 nM product peptide as 100 and immediately terminated reaction as 0. Reaction conditions for 2-OG Km determinations were the same as above except that the enzyme concentration was 6 nM KDM5AFL, 12 nM KDM5A12–797 or 4 nM KDM5AΔPHD1, and 2-OG was titrated between 0.1 and 50 μM. Reactions were quenched with a half-volume addition of stop buffer (50 mM Tris-HCl, pH 7.5, 0.01% Triton X-100, 233 mM KF, 2.3 mM EDTA) after 2–8 min at ambient temperature. The TR-FRET ratio was measured 1 h after a quarter-volume of detection (2 nM H3K4me2-Eu(K), 100 nM SA-XL665 in stop buffer) was added to the quenched reactions. Background signals of immediately terminated reactions at each 2-OG concentration were subtracted from every reaction time. Reaction rates (min−1) were calculated from linear fits, and the Michaelis–Menton model was used to determine Km and Vmax using Prism 6.0 (GraphPad). All kinetic parameters and activity measurements are presented as an average of three independent experi-ments with s.d. This assay was used for the determination of kinetic parameters of the crystallographic constructs described in Supplementary Table 8.

Construct design for structural studies. We analyzed the expression of KDM5A constructs of different lengths and domain combinations in Sf9 insect cells. All first-round expression constructs minimally contained the catalytic JmjN and JmjC, ARID and PHD1 domains. Reading frames (Supplementary Table 5) were cloned into both N-terminal His6 and C-terminal His6-tag containing vectors (BiNTH and BiCTH). Our first round of clones yielded expressed frag-ments (12–738, 12–797, 12–861 and 12–1,660) and non-expressed fragments (12–625, 12–671 and 12–1,218). We found that N- or C-terminal tagging did not affect expression, except in the 12–861 fragment, which expressed well when C-terminally, but not N-terminally, tagged. Purification screens of expressed constructs further stratified the constructs into monomeric, homo-geneous constructs (12–738, 12–797 and 12–1,660) and an aggregated, poorly behaved construct (12–861). Several constructs resulting in soluble, mono-meric protein were taken into crystallization experiments, but only constructs with external boundaries 12–797 crystallized.

Protein expression, purification and crystallization. Human KDM5A12–797 was subcloned into the pAcGP67 vector (BD Biosciences). The transfer vec-tor was cotransfected with BestBac linearized viral DNA (Expression Systems) into Sf9 cells using Cellfectin (Invitrogen) to produce recombinant baculovi-rus. The protein was expressed in Sf9 cells as N-terminally His6-tagged protein with tag cleavable by TEV. A 22L Wave bioreactor was inoculated with Sf9 at 1 × 106 cells/mL in serum-free ESF921 (Expression Systems). The cells were lysed using microfluidizer (Microfuidics) in buffer containing 25 mM Tris, pH 7.5, 200 mM NaCl, 10% glycerol, 0.5 mM TCEP, and Complete EDTA-free pro-tease inhibitors cocktail (Roche). The cell lysate was cleared by centrifugation at 40,000g for 1 h. The supernatant was loaded onto Ni-NTA agarose beads (Qiagen). The beads were washed using the protein buffer (25 mM Tris, pH 7.5, 200 mM NaCl, 10% glycerol, 0.5 mM TCEP) containing 20 mM imidazole, and the bound protein was eluted using the same buffer with 100 mM imidazole. The samples of KDM5A12–797 were treated with TEV and loaded onto HiLoad gel-filtration column Superdex 200 16/600 (GE Healthcare Life Sciences). The protein was eluted as a single peak. The fractions containing the pure sample were concentrated using centrifuge filters (Amicon). KDM5A12–797 protein at

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10 mg/mL was cocrystallized with 200 μM CPI-455 in the presence of 20% PEG3350, 0.1 M HEPES, pH 7.3, and 12% glycerol by vapor diffusion. Prior to data collection, all crystals were flash-cooled in liquid nitrogen.

Structure determination and refinement. Data were collected at either the Canadian Light Source CMCF-08ID or Advanced Light Source beamline 5.0.2 (Supplementary Tables 6 and 7) at a wavelength of 0.97949 Å (peak for SeMet absorption) and were integrated and scaled with HKL2000 (ref. 43). The Rmerge for the native data set in the highest resolution bin of data was elevated; how-ever, analysis of other data quality assessments such as CC1/2 (Supplementary Table 7) demonstrated that those data remained reliable for inclusion in the refinement. The crystal structure of KDM5A was determined by a combina-tion of molecular replacement and single-wavelength anomalous dispersion (MR-SAD) using protein labeled with selenomethionine during expression. The CPI-455–KDM5A12–797 complex crystal was of space group P3121, with a single molecule in the asymmetric unit and the following cell dimensions: a = b = 159.3 Å, c = 92.5 Å, α = β = 90°, γ = 120°. We obtained the MR solution for KDM5A12–797 using a search model derived from an internal structure of KDM4C in which large loops had been truncated (approximately one-fourth of the scattering mass of the crystal). An anomalous difference Fourier peak search combined with the MR solution (PHASER-EP and Autosol/RESOLVE44) generated 14 peaks and a starting figure of merit of 0.47 (Supplementary Table 6). The model was adjusted in Coot45 and used to solve subsequent structures by MR, including an additional 3.14-Å CPI-455 complex structure that is described here. Refinement of this second CPI-455 structure was con-ducted in Phenix44 and resulted in an Rfree of 23.8% with good stereochemis-try (Supplementary Tables 6 and 7). The final model contained amino acids 12–183 and 361–785, with remaining amino acid residues disordered. Relatively strong but discontinuous difference electron density for a short stretch of amino acids (approximately ten residues) beyond residue 183 exists but was not sufficient to convincingly set the registration of the chain. Ten residues of polyalanine have been fitted to this section and assigned the amino acid type of UNK and an arbitrary residue number of 219–228, in compliance with guide-lines from the RCSB staff. This short segment has been omitted from figures for clarity and would not include the missing PHD1 domain. The structure has 92.9% of its residues in the favored region of the Ramachandran plot and five

residues in ‘disallowed’ regions. Three of these higher-energy conformations occur at regions where insertions or deletions of the KDM5A sequence rela-tive to that of the KDM5B sequence exist or where the KDM5B sequence has a proline substitution, potentially suggesting that the length and/or sequence changes necessitate these conformations. Higher-resolution structures of KDM5A would be required to further investigate that possibility. Electron density for ligand was unambiguous (Supplementary Fig. 2a). The active-site metal and the metal ions in the zinc finger were assigned on the basis of induc-tively coupled plasma mass spectrometry analysis of the metals present in the protein samples. No crystals were obtained when exogenous iron was added to the protein buffer.

Database deposition. Coordinates and structure factors for the crystal struc-ture of KDM5A in complex with CPI-455 have been deposited at the RCSB PDB under the accession code 5CEH.

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