TAK-243 | Glyr Signal

A small-molecule inhibitor of the ubiquitin activating enzyme for cancer treatment

Marc L Hyer1–3, Michael A Milhollen1,3, Jeff Ciavarri1, Paul Fleming1, Tary Traore1, Darshan Sappal1, Jessica Huck1, Judy Shi1, James Gavin1, Jim Brownell1, Yu Yang1, Bradley Stringer1, Robert Griffin1, Frank Bruzzese1, Teresa Soucy1, Jennifer Duffy1, Claudia Rabino1, Jessica Riceberg1, Kara Hoar1,ImageAnya Lublinsky1, Saurabh Menon1, Michael Sintchak1, Nancy Bump1, Sai M Pulukuri1, Steve Langston1, Stephen Tirrell1, Mike Kuranda1, Petter Veiby1, John Newcomb1, Ping Li1, Jing Tao Wu1, Josh Powe1, Lawrence R Dick1 , Paul Greenspan1, Katherine Galvin1, Mark Manfredi1, Chris Claiborne1, Benjamin S Amidon1 & Neil F Bence1,2

The ubiquitin–proteasome system (UPS) comprises a network of enzymes that is responsible for maintaining cellular protein homeostasis. The therapeutic potential of this pathway has been validated by the clinical successes of a number of UPS modulators, including proteasome inhibitors and immunomodulatory imide drugs (IMiDs). Here we identified TAK-243 (formerly known as MLN7243) as a potent, mechanism-based small-molecule inhibitor of the ubiquitin activating enzyme (UAE), the primary mammalian E1 enzyme that regulates the ubiquitin conjugation cascade. TAK-243 treatment caused depletion of cellular ubiquitin conjugates, resulting in disruption of signaling events, induction of proteotoxic stress, and impairment of cell cycle progression and DNA damage repair pathways. TAK-243 treatment caused death of cancer cells and, in primary human xenograft studies, demonstrated antitumor activity at tolerated doses. Due to its specificity and potency, TAK-243 allows for interrogation of ubiquitin biology and for assessment of UAE inhibition as a new approach for cancer treatment.

Ubiquitin conjugation in mammals is initiated by two key enzymes, the ubiquitin activating enzymes UAE (also known as UBE1) and UBA6, which are collectively referred to as E1 enzymes. UAE (encoded by the UBA1 gene) is responsible for charging an estimated
>99% of cellular ubiquitin, whereas UBA6 is responsible for charging<1% of ubiquitin. UAE catalyzes ubiquitin-charging of all (~35) E2 cellular ubiquitin-conjugating enzymes, except for the E2 USE1, whose charging is catalyzed by UBA6 (ref. 1). Ubiquitin-charged E2 enzymes cooperate with cellular E3 ligases (for example, cullin, Ring, HECT, RBR or U-box) to direct specific cellular target protein ubiquitylation modifications, which are classified as monomeric or polyubiquitin; moreover, polyubiquitin chains can be of the Lys11 (K11), Lys29 (K29), Lys48 (K48) or Lys63 (K63) type. These varied ubiquitin modifications are read by ubiquitin-binding proteins and can dictate outcomes in which the target proteins are either degraded or not. For example, K48-linked polyubiquitin is associated with proteasome-mediated degradation, K63-linked polyubiquitin can mediate autophagy and signal transduction, polyubiquitylation has been shown to be important for signal transduction mediated by the transcription factor NF-kB2,3, mono-ubiquitylation of histones can alter gene regulation, and mono-ubiquitylation of surface receptors can modulate their internalization and lysosomal proteolysis.

The clinical success of the proteasome inhibitor bortezomib4 has piqued interest in targeting other components of the UPS for cancer therapy. Although the structural and mechanistic diversity of UPS enzymes has presented challenges for the small-molecule interrogation of E1, E2, E3 and deubiquitinating (DUB) enzymes in cancer biology, progress is emerging with ongoing clinical evaluation of pevonedistat (also known as TAK-924 and MLN4924; an inhibitor of the E1 for the ubiquitin-like molecule NEDD8)5, second-generation IMiDs, inhibitors of the E3 ligases for inhibitor of apoptosis protein (IAP) and murine double minute 2 (MDM2), and the valosin-containing protein (VCP) inhibitor CB-5083 (refs. 6–9).
UBA1 is an essential gene in yeast10,11. Although no data has been published on the effects of genetic ablation of Uba1 in mice, it is likely to cause lethality in this species as well. Here we report iden- tification of TAK-243 (MLN7243), a first-in-class inhibitor of UAE. TAK-243 is a mechanism-based inhibitor that potently inhibits UAE via formation of a TAK-243–ubiquitin adduct. Treatment of cells in vitro with TAK-243 led to loss of cellular ubiquitin conjugates, resulting in defective ubiquitin-dependent protein turnover and sig- naling, impaired cell cycle progression and defective DNA repair, increased proteotoxic stress, and ultimately cancer cell death. TAK- 243 treatment of tumor cells in vivo caused a dramatic reduction of1Takeda Pharmaceuticals Inc., Cambridge, Massachusetts, USA. 2Present addresses: Agios Pharmaceuticals, Cambridge, Massachusetts, USA (M.L.H.) and Nurix, Inc., San Francisco, California, USA (N.F.B.). 3These authors contributed equally to this work. Correspondence should be addressed to M.A.M. ([email protected]).
Received 30 September 2016; accepted 19 December 2017; published online 15 January 2018; doi:10.1038/nm.4474cellular polyubiquitination and induced pronounced antitumor activ- ity in mice bearing human xenograft tumors.

RESULTS
TAK-243 forms a TAK-243–ubiquitin adduct and potently inhibits UAE in vitro
We undertook a medicinal-chemistry-based effort to generate small- molecule inhibitors of UAE, which yielded >700 potential candidates. We then screened these candidates using an in vitro biochemical assay that measured UAE-mediated transfer of ubiquitin to an E2 substrate (UBCH10) and identified a potent, mechanism-based UAE inhibitor, TAK-243 (Fig. 1a). TAK-243 had a half-maximal inhibitory concentration (IC50) value of 1 ± 0.2 nM (Fig. 1b) in the UBCH10 E2 thioester assay. Using similar biochemical assays, we found that TAK-243 had weaker inhibitory activity against other closely related E1 ubiquitin-like activating enzymes such as Fat10- activating enzyme (UBA6; 7 ± 3 nM), NEDD8-activating enzyme (NAE; 28 ± 11 nM), SUMO-activating enzyme (SAE; 850 ± 180 nM), ISG15-activating enzyme (UBA7; 5,300 ± 2,100 nM) and autophagy- activating enzyme (ATG7; >10,000 nM) than it did against UAE. Consistent with the concept that TAK-243 inhibits UAE by using a
substrate-assisted mechanism of action similar to that described previously for the small-molecule NAE inhibitor pevonedistat5, X-ray crystallographic studies revealed that TAK-243 was bound to a site of UAE (humanized yeast UAE) normally occupied by AMP (Fig. 1c). Notably, a continuous electron-density unit was observed to con- nect the C terminus of ubiquitin to the sulfamate moiety of TAK-243 (Fig. 1c and Supplementary Table 1), indicating the presence of a covalent TAK-243–ubiquitin adduct. We attempted to crystallize human UAE bound to TAK-243; however, crystals were difficult to obtain, and they diffracted to a lower resolution as compared to that with a humanized yeast UAE version, which has recently been used by others12.
Additional biochemical assays (Supplementary Fig. 1) dem- onstrated that TAK-243 is a time-dependent human UAE inhibitor (Supplementary Fig. 1a,b) that has a mechanism of action consist- ent with a substrate-assisted mechanism previously described for the small-molecule NAE inhibitor pevonedistat5. The TAK-243–ubiquitin adduct (Fig. 1c), once formed, remained tightly bound to UAE and blocked UAE catalytic activity (Supplementary Fig. 1a). A transthi- olation assay showed that TAK-243 inhibited UAE from transfer- ring ubiquitin to an E2 enzyme (Supplementary Fig. 1c). TAK-243 showed high selectivity, as indicated by minimal inhibitory activity

TAK-243 is a mechanism-based, cell-active inhibitor of UAE. (a) Chemical structure of the adenosine sulfamate UAE inhibitor TAK-243.
(b) Dose-response inhibition profiles of TAK-243 for the E1 enzymes UAE, UBA6, NAE, SAE, UBA7 and ATG7. Data represent the mean ± s.d. of
n = 2 experiments run with duplicate samples. (c) Structure, as shown using a ribbon diagram (left) and an electron density map (right), of the TAK- 243–ubiquitin adduct species in which TAK-243 forms a covalent bond with the C terminus of ubiquitin. The electron density map shows that the TAK- 243–ubiquitin adduct occupies the adenylate (AMP)-binding site of UAE. (d) Representative western blot analysis (of n = 2 independent experiments) for the response of HCT-116 cells to different concentrations of TAK-243. The status of ubiquitin and ubiquitin-like protein charging on the E1 and E2 enzymes UBCH10 (UAE-specific ubiquitin E2), USE1 (UBA6-specific ubiquitin E2), UBC12 (NEDD8 E2), UBC9 (SUMO E2) and ATG7 (autophagy UBL E1) was assessed.

TAK-243 inhibits cellular ubiquitin conjugation, which leads to substrate stabilization, cell cycle arrest, ER stress and an impaired DNA damage response. (a) Representative western blot analysis (of n = 2 independent experiments) showing dose response and time course of TAK-243 target engagement in HCT-116 cells, as assessed by immunoblotting for the TAK-243–ubiquitin adduct (MIL90), polyubiquitin (polyUb), ubiquitylated histone H2B, c-Jun, c-Myc, MCL1, XIAP and p53. Tubulin was used as a loading control; UAE (UBE1) levels are also shown. (b) FACS analysis showing DNA content in HCT-116 cells at 24 h after TAK-243 treatment. FL2 represents PI+ cells. The NAE inhibitor pevonedistat was used for comparison.
Plots are representative of n = 2 independent experiments. (c) Representative western blot analysis (of n = 2 independent experiments) showing dose response and time course of the effects of TAK-243 on the UPR and apoptosis in HCT-116 cells, as assessed by immunoblotting for BIP, ATF6, PERK, phosphorylated ERN1 (p-IRE1a), XBP1s, phosphorylated eIF2a (p-eIF2a), ATF4, CHOP and GADD34, cleaved caspase-3 and cleaved PARP. The PERK-specific antibody recognizes phosphorylated and unphosphorylated PERK; the high-molecular-weight band stained by anti-PERK corresponds to phosphorylated PERK. Tubulin was used as a loading control. (d) E2–ubiquitin enzyme charging in HCT-116 cells at 8 h after TAK-243 treatment, as
assessed by western blotting. The following UAE-specific E2 enzymes were evaluated: UBE2A, UBE2B, UBE2T, PCNA and FANCD2. UAE was included as a loading control. Western blots are representative of n = 2 independent experiments. (e) Representative images (of n = 2 independent experiments) showing DNA damage repair, as assessed by COMET assays, in Calu-6 cells that were treated with UV radiation and then treated with DMSO or TAK-243 (1 mM) for 0 or 6 h. Scale bar, 10 mm. See Supplementary Figure 7 for quantification and dose response.
© 2018 Nature America, Inc., part of Springer Nature.
All rights reserved.

in a panel of kinase and receptor assays, as well as on human car- bonic anhydrase type I and type II (Supplementary Table 2). Taken together, these data indicate that UAE and UBA6, the ubiquitin E1 enzymes, are the primary biochemical targets of TAK-243.
Next we characterized the selectivity of TAK-243 in a well-character- ized xenograft-amenable HCT-116 colon cancer cell line using a panel of ubiquitin-like (UBL)-thioester charging assays. These assays meas- ure the efficiency of an E1 enzyme, such as UAE, to transfer ubiquitin or a UBL-like moiety to an E2 enzyme. Consistent with the in vitro results using purified UAE, TAK-243 showed strong selectivity over the Sumo and autophagy UBL pathways, as indicated by the negligible effect of TAK-243 treatment on the UBC9SUMO:UBC9 charging ratio
and by a slight increase in the ATG7UBL:ATG7 charging ratio (Fig. 1d). The latter effect may reflect induction of autophagy due to activation of the integrated stress response. TAK-243 was tenfold more selec- tive against UAE than against NAE, as indicated by its effects on the UBCH10Ub and UBC12NEDD8 charging ratios. TAK-243 was showed equally potent inhibition of the two E1 enzymes capable of activating ubiquitin (UBA6 and UAE), as indicated by comparable decreases in levels of charged USE1 (USE1Ub) and UBCH10 (UBCH10Ub), using the cell-based E2~UBL thioester assay (Fig. 1d).

We also generated a UBA6-specific small-molecule inhibitor (US patent US9593121B2; section 067, figure I-01)13 as a tool to inter- rogate the consequences of selectively inhibiting UBA6 activity.
© 2018 Nature America, Inc., part of Springer Nature. All rights reserved.

Table 1 Anti-proliferative EC50 values of TAK-243 in cell lines
Cell line Origin TAK-243 (mM)
HCT116 Colon 0.020 ± 0.01 (16)
HT29 Colon 0.308 ± 0.08 (4)
LOVO Colon 0.450 ± 0.14 (3)
SW480 Colon 0.044 ± 0.01 (3)
SW48 Colon 0.056 ± 0.02 (10)
H460 Lung 1.31 ± 0.19 (9)
Calu6 Lung 0.174 ± 0.17 (7)
A549 Lung 0.816 ± 0.38 (6)
NCI-H520 Lung 0.013 ± 0.01 (4)
NCI-H69 Lung 0.011 ± 0.01 (3)
NCI-H82 Lung 0.006 ± 0.00 (3)
MDA-MB-231 Breast 0.172 ± 0.14 (4)
HCC1954 Breast 0.123 ± 0.03 (4)
A375 Melanoma 0.381 ± 0.28 (10)
WM266.4 Melanoma 0.063 ± 0.01 (3)
MIA-PACA-2 Pancreatic 0.045 ± 0.02 (3)
HL60 Leukemia 0.017 ± 0.01 (6)
MV-411 Leukemia 0.015 ± 0.01 (4)
THP-1 Leukemia 0.019 ± 0.01 (4)
OCI-M2 Leukemia 0.132 ± 0.04 (4)
MM1.S Multiple myeloma 0.012 ± 0.01 (4)
RPMI-8226 Multiple myeloma 1.099 ± 0.22 (4)
NCI-H929 Multiple myeloma 0.109 ± 0.01 (3)
WSU-DLCL2 Lymphoma 0.016 ± 0.01 (5)
MCF10A cycling Breast epithelial 0.033 ± 0.01 (3)
MCF10A non-cycling Breast epithelial 0.021 ± 0.01 (3)
Normal dermal fibroblast Human fibroblast 0.934 ± 0.05 (2)
UBA6 (WT) MEFs Mouse embryonic fibroblast 0.792 ± 0.39 (3)
UBA6-null MEFs Mouse embryonic fibroblast 2.580 ± 1.06 (3)
UBE1 overexpression HCT116 Colon 0.021 ± 0.01 (3)
UBE1 vector control HCT116 Colon 0.036 ± 0.02 (3)

The indicated tumor cell lines, as well as normal dermal human fibroblasts and MEFs that were either wild type (WT) or a knockout for UBA6, were treated as described in the Online Methods, and the concentration at half maximal viability (EC50) value ± s.d. is reported. UBE1-overexpressing HCT-116 cells are further described in Supplemen- tary Figure 2b. Numbers in parentheses represent the number of times the assay was performed.

Treatment of HCT-116 cells with this UBA6 inhibitor had negligible effects on the levels of bulk polyubiquitin chains, NEDDylated cul- lins and high-molecular-weight SUMOylated species, despite showing robust, specific inhibition of the UBA6-specific charging of the E2 enzyme USE1 (Supplementary Fig. 2a–d).

To further characterize TAK-243, we evaluated its effects on a panel of UAE-specific E2 enzymes (UBCH10, UBC1, UBC2, UBC3, UBC5, UBC13, UBE2T, UBE2A and UBE2B) using the cellular E2~UBL thioester assay (Supplementary Fig. 2e,f). In these experi- ments, HCT-116 colon cancer cells and WSU-DLCL2 lymphoma cells were treated with TAK-243, and western blotting analysis was used to measure ubiquitin-charged E2s. TAK-243 treatment resulted in a uniform, dose-dependent loss of ubiquitin-charged E2~UBL thioesters (Supplementary Fig. 2e,f), consistent with the conclu- sion that TAK-243 treatment leads to uniform disruption of cellular ubiquitin-conjugation events. Moreover, TAK-243 treatment reduced the global levels of K48-linked polyubiquitylated chains in HCT- 116 cells in a dose-dependent manner (Supplementary Fig. 2g).

As expected, TAK-243 modulated E2 thioester charging and K48-linkedchain formation at comparable half-maximal effective concentration (EC50) values.TAK-243 inhibits the turnover of short-lived proteins and disrupts cell cycle progressionBecause ubiquitin is known to target proteins for degradation, we evaluated the effect of UAE inhibition on the turnover of short- lived proteins by using a cellular assay that monitors the release of 35S-methionine from metabolically labeled proteins. Side-by-side controls in this experiment included inhibitors of the proteasome (ixazomib), NAE (pevonedistat) and UBA6 (UBA6i) (Supplementary Fig. 3). TAK-243 treatment resulted in protein turnover inhibition similar to that observed with a proteasome inhibitor. In contrast, UBA6 inhibition had no effect on protein turnover, and inhibition of NAE had a modest effect (20%) (Supplementary Fig. 3), consist- ent with previous work14. Inhibition of the proteasome and UAE decreased protein turnover to similar extents, suggesting the bulk of proteasome-dependent protein turnover is ubiquitin dependent (Supplementary Fig. 3).

We further explored the effect of TAK-243 on protein turnover by measuring the effects of TAK-243 treatment on the stability of specific short-lived oncoproteins and tumor suppressors (c-Jun, c- Myc, MCL1, XIAP and p53), which are dependent on ubiquitin for degradation. The solid tumor cell line, HCT-116, was selected for this experiment because, unlike the proteasome inhibitor bortezomib, TAK-243 demonstrated broad antitumor activity in models of solid and hematological tumors (see below). An antibody (MIL90) raised against the TAK-243–ubiquitin adduct species was used to detect adduct formation and target engagement in cells12 (Supplementary Fig. 4). At the earliest time point examined (1 h), TAK-243–ubiquitin adduct formation was clearly evident in HCT-116 cells (Fig. 2a). In addition, downstream UAE pathway inhibition by TAK-243 was evi- dent, as shown by a dose- and time-dependent loss of both polyubiq- uitin chains and mono-ubiquitylated histone H2B; however, TAK-243 treatment did not affect UAE (UBE1) protein levels (Fig. 2a). TAK- 243 treatment also caused accumulation of short-lived proteins such as c-Jun, c-Myc, MCL1 and p53 (Fig. 2a), consistent with the concept that loss of polyubiquitylated chains impairs the targeting of proteins for proteasomal degradation. For example, TAK-243 treatment led to accumulation of both p53 and c-Myc, both of which have been reported to have half-lives of <60 min in HCT-116 cells15,16.

Ubiquitin is known to have an essential role in cell cycle regula- tion15. To examine the effect of UAE inhibition on cell cycle regula- tion, we used flow cytometric analysis to measure the DNA content in TAK-243-treated HCT-116 cells. TAK-243 treatment resulted in a complete G2/M arrest at concentrations near its EC50 value (0.05 mM); however, at higher concentrations (>EC90, 1 mM), TAK-243 treat- ment resulted in arrest at both the G1 and G2/M phases of the cell cycle, which was suggestive of the importance of ubiquitylation on proteins that are critical for proper passage through the cell cycle (Fig. 2b). Protein levels of the cell cycle markers cyclin B and cyclin D1 and of the cyclin-dependent kinase (CDK) inhibitors p21 and p27 correlated with the cell cycle DNA profiles observed in TAK- 243-treated cells (Supplementary Fig. 5a). Notably, the dominant cell cycle phenotype observed after TAK-243 treatment was different than that after treatment with an NAE inhibitor but was more similar to that after treatment with a proteasome inhibitor (ixazomib)16. In particular, NAE inhibition (using pevonedistat) has been reported to induce an S-phase re-replication phenotype (DNA content ³ 4N) through stabilization and dysregulation of chromatin licensing and© 2018 Nature America, Inc., part of Springer Nature. All rights reserved.

DNA replication factor 1 (CDT1) turnover14,17; in contrast, a re- replication phenotype was not observed in TAK-243-treated cells, as no cell population with DNA content ³ 4N was detected (Fig. 2b). Unlike pevonedistat, TAK-243 treatment stabilized both CDT1 and its endogenous inhibitor geminin (Supplementary Fig. 5b), which would presumably prevent re-replication. Additionally, unlike pev- onedistat11,18, TAK-243 treatment did not cause loss of the mitotic cell cycle protein histone H3 that is phosphorylated on Ser10 (pH3 (Ser10)) (Supplementary Fig. 5a).
TAK-243 induces irresolvable endoplasmic reticulum stress Impaired ubiquitylation is anticipated to profoundly affect protein quality control, especially within the endoplasmic reticulum (ER). Ubiquitin is known to be important for proper retro-translocation of misfolded proteins from the ER to the cytosol, and thus, unlike proteasome inhibition, inhibition of ubiquitylation by TAK-243 would be expected to enhance accumulation of misfolded proteins within the ER membrane and lumen19. Consistent with this hypoth- esis, TAK-243 treatment of HCT-116 cells led to robust activation of the unfolded protein response (UPR), as evidenced by increased phosphorylation of the kinase PERK (PERK mobility shift) and of the protein endoplasmic reticulum to nucleus signaling 1 (ERN1; also known as IRE-1a) as well as accumulation of ‘activating transcription factor 6’ (ATF6) (Fig. 2c). Moreover, TAK-243 treatment induced rapid expansion of the ER surface area and led to hyper-vesiculization within this organelle (Supplementary Fig. 6), strongly suggesting the induction of ER stress. TAK-243 treatment also led to activa- tion of the downstream ER stress signals XBP1s and phosphorylated eIF2a, and ATF4 (suggestive of prolonged ER stress), induced acti- vation of the mediators of ER-stress-induced apoptosis CHOP and GADD34, and ultimately resulted in cellular apoptosis, as indicated by the accumulation of cleaved PARP (poly(ADP-ribose) polymerase
1) and cleaved caspase-3 (Fig. 2c).

TAK-243 treatment alters DNA damage repairUbiquitylation is critical for coordinated regulation of the DNA damage response18,20,21. TAK-243 treatment led to impairment in the charging of multiple E2 enzymes associated with DNA damage repair, including UBC13, UBE2T, UBE2A and UBE2B (Fig. 2d and Supplementary Fig. 2e). Therefore TAK-243 treatment would be expected to impair multiple DNA repair pathways, including homol- ogous recombination, interstrand cross-link repair and post-repli- cative repair. Consistent with its effects on the charging of UBE2T, UBE2A and UBE2B, TAK-243 inhibited the mono-ubiquitylation of PCNA and FANCD2, two key substrates for interstrand cross-linking and post-replicative repair, with no effects on UBE1 protein levels (Fig. 2d). To test for a functional consequence of TAK-243 on DNA repair, we directly assessed DNA damage following ultraviolet (UV) irradiation using the COMET assay22. The lung carcinoma cell line Calu-6 was used for improved COMET assay reproducibility. Calu-6 cells were pretreated for 1 h with 1 mM TAK-243 or DMSO (control) and then exposed to UV irradiation (20 J/m2). Immediately following irradiation (time t = 0), damaged DNA (comet tails) was observed in both the DMSO-treated and TAK-243-treated cells (Fig. 2e). At 6 h after UV irradiation, there were far fewer comet tails in cells that were treated with DMSO (control), suggesting that the DNA damage was repaired; however, TAK-243-treated cells showed persistent, unre- solved DNA damage (Fig. 2e and Supplementary Fig. 7). TAK-243 treatment alone (in the absence of UV irradiation) did not induce DNA damage (Supplementary Fig. 7).

These results are consistentwith previous work linking ubiquitin to DNA damage repair23 and suggest that TAK-243 could enhance the antitumor activity of clini- cally validated DNA damaging agents. To test this hypothesis in vivo, we combined TAK-243 treatment with beam-focused radiation in two primary xenograft (PDX) models of cancer (breast and non-small-cell lung cancer (NSCLC)). Consistent with our hypothesis, combined treatment led to complete tumor responses in both of these models (Supplementary Fig. 8).

TAK-243 has anti-proliferative activity in human cancer cells Given our findings that UAE inhibition leads to proteotoxic stress (ER stress and UPR), impaired cell cycle progression and inhibition of DNA repair, we reasoned that TAK-243 would display a broad anti-prolif- erative activity against cancer cells. We assessed the anti-proliferative effect of TAK-243 on a panel of cell lines derived from hematologic and solid tumors. TAK-243 treatment induced complete cell killing of all of the lines tested, with variable EC50 values that ranged from
0.006 mM to 1.31 mM (Table 1). Many of the tumor cell lines demon-strated EC50 values in the double-digit nanomolar range.

In contrast, TAK-243 showed weaker cytotoxic effects (1 mM) on normal human dermal fibroblasts, suggesting that a therapeutic window might exist for TAK-243 treatment in vivo. The anti-proliferative effects of TAK- 243 are likely to be driven by apoptosis, as apoptotic cell death mark- ers (cleaved PARP and cleaved caspase-3) were detectable following treatment (Fig. 2c). Of note, TAK-243 demonstrated substantial anti- proliferative effects in vitro in multiple-myeloma-derived cell lines (Table 1), an indication for which proteosome inhibitors have shown clinical benefit. For example, bortezomib and TAK-243 had similar EC50 values in vitro for MM1.S multiple-myeloma cells (0.011 ± 0.001
mM (n = 4) and 0.012 ± 0.001 mM (n = 4), respectively). The potency of TAK-243 showed no correlation with UBA1 mRNA levels across a panel of tumor cell lines and was not significantly affected by UAE overexpression (Table 1 and Supplementary Fig. 9a,b). A lack of correlation between potency and target levels is not uncommon, as similar observations have been noted for other drugs that target the kinase PLK or MEK, the proto-oncoprotein b-RAF and NAE24–27.

Moreover, TAK-243 potency did not correlate with proliferation rates of the tumor cell lines tested (Supplementary Fig. 9c).
In addition to inhibiting UAE, TAK-243 was also found to inhibit UBA6 in cells (Fig. 1d). We therefore attempted to tease apart the contribution of UBA6 inhibition on cell viability. UBA6-knockout cells were used to test the hypothesis that lack of UBA6 confers sen- sitivity of mouse embryonic fibroblasts (MEFs) to TAK-243. Viability experiments comparing TAK-243-treated wild-type (WT) and UBA6- knockout MEFs indicated that, if anything, UBA6-knockout cells were more resistant to TAK-243 treatment than WT MEFs (approximately threefold decrease in sensitivity) (Table 1). Additionally, anti-pro- liferative effects of UBA6-specific inhibitors were observed only at concentrations 10- to 100-fold higher than that required for TAK-243 in the cell lines tested (data not shown). Collectively these findings support the idea that TAK-243 exerts its cell viability effect through inhibition of UAE.

Pharmacokinetic and pharmacodynamic analysis of TAK-243 in tumor bearing mice
We characterized the pharmacokinetic (PK) parameters of TAK-243 in immunocompromised CB-17 SCID mice that bore a subcutane- ous diffuse, large B cell lymphoma (WSU-DLCL2) tumor following an acute, intravenously administered dose of TAK-243. Both plasma and WSU-DLCL2 tumor tissues from these mice were analyzed forexposure to TAK-243. In plasma, the TAK-243 parent compound was characterized by a high clearance (CL) rate (3.99 to 4.99 liter per h per kg body weight (liter/h/kg)) and a short terminal half-life (t1/2) (0.2–0.4 h) (Supplementary Fig. 10a). TAK-243 rapidly forms a TAK- 243–ubiquitin adduct, and our PK measurements did not account for TAK-243 that was sequestered as a ubiquitin adduct. Despite the high plasma clearance rate observed for TAK-243, the drug showed a high volume of distribution at steady state (Vss) (1.13–1.74 liter/h/kg) with a 5.8- to 8-fold higher area-under-the-curve (AUC) drug exposure in tumor tissue relative to that in plasma, and a prolonged tumor t1/2 of

we next evaluated TAK-243 target engagement with UAE and down- stream pathway modulation in tumor samples. We developed immu- nohistochemistry (IHC) assays to analyze several pharmacodynamic (PD) biomarkers associated with UAE inhibition. The monoclonal antibody raised against the TAK-243–ubiquitin adduct (MIL90) was used to monitor UAE target engagement in tumors. Downstream pathway inhibition was evaluated using several biomarkers; antibodies were used to detect both polyubiquitylated and mono-ubiquitylated proteins (using the FK2 antibody, which detects all cellular forms of ubiquitin conjugates), mono-ubiquitylated histone H2B and cleaved caspase-3 (as a measure of apoptosis). Mice bearing either WSU- DLCL2 lymphoma or PHTX-132Lu (primary NSCLC) xenograft tumor tissues were dosed using a single intravenous injection of TAK- 243 at its maximum tolerated dose. Both UAE target engagement, as assessed by the presence of the TAK-243–ubiquitin adduct, and downstream pathway inhibition were clearly evident in tumor tis- sue (Fig. 3a).

Following a single dose, TAK-243 induced a rapid and prolonged PD response (Fig. 3b,c), consistent with pronounced UAE inhibition in tumor tissue. Of the PD biomarkers evaluated, adduct formation was detected most sensitively, and the adduct could be© 2018 Nature America, Inc., part of Springer Nature. All rights reserveddetected at very low TAK-243 doses (3 mg per kg body weight (mg/kg) was the lowest dose examined) that were insufficient to modulate downstream PD readouts (polyubiquitin or mono-ubiquitylated histone H2B) or antitumor efficacy (data not shown). The adduct could be detected 0.5–1.0 h before downstream biomarker modula- tion, a lag which could be due to a combination of differences in assay sensitivity, de novo UAE synthesis and drug washout in the tumor over time. Of note, at its maximally tolerated dose, TAK-243 showed negligible inhibition of NAE-dependent neddylated cullin levels in HCT-116 xenografts, despite profound inhibition of polyubiquitin formation and ubiquitylated histone H2B levels (Supplementary Fig. 11a,b). These data indicate TAK-243 profoundly affects down- stream UAE biomarkers while having little-to-no impact on NAE- associated biomarkers in vivo.

TAK-243 has UAE-specific antitumor efficacy in vivo
The antitumor activity of TAK-243 was determined with a panel of human-patient-derived and cell-line-derived xenograft (PDX and CDX, respectively) tumor models that represented both solid and hematological cancers. We established subcutaneous tumors in mice using the following CDX models: WSU-DLCL2 (diffuse, large B cell lymphoma), HCT-116 (colon carcinoma), THP-1 (acute myeloid leukemia), CWR22 (prostate cancer), Calu-6 (lung, non-small-cell adenocarcinoma (NSCLC)), HCC70 (triple-negative breast cancer) and MM1.S (multiple myeloma), as well as the following human pri- mary PDX models: PHTX-24c (colon cancer), PHTX-132Lu (lung, NSCLC), PHTX-55B (triple-negative breast cancer), PHTX-235O (ovarian cancer), and HNM626 (neck cancer). Tumor-bearing miceFigure 3 TAK-243 induces a pharmacodynamic (PD) response in xenograft tumors. Mice bearing WSU-DLCL2 lymphoma or PHTX-132Lu primary human NSCLC tumors were treated with a single intravenous dose of TAK-243 (25 mg/kg) or vehicle control. (a) Representative IHC images (of n = 3 mice per time point) of the TAK-243–ubiquitin adduct (4 h post-dosing, total cellular ubiquitin conjugates (FK2 antibody; 8 h after dosing), monoubiquitylated H2B (anti-H2BUb; 8 h after dosing), and apoptosis (cleaved caspase-3; 24 h after dosing) in tumor tissue. Scale bar, 100 mm. (b) Quantification of the area that stained positive for presence of the TAK-243–ubiquitin adduct in WSU-DLCL2 (blue line) and PHTX-132Lu (red line) tumor tissue over time. (c) Quantificationof the area that stained positive for polyubiquitin conjugates (red line) and H2BUb (black dashed line) in WSU-DLCL2 (top) and PHTX-132Lu (bottom) tumor tissue over time. For b,c, data points indicate mean ±
s.e.m. (n = 3 tumors/time point)were dosed for 3 weeks with TAK-243,which was administered intra- venously on a twice-per-week schedule (for example, Monday and Thursday; denoted BIW), and tumor growth and animal body weight were monitored. TAK-243 treatment induced a marked and robust antitumor activity response in all of the models examined (Fig. 4, Table 2 and Supplementary Fig. 12a). The human cancer cell lines that were treated with TAK-243 both in vitro and in vivo retained a similar rank order in sensitivity. TAK-243 inhibited both mouse and human UAE, as both TAK-243–ubiquitin adduct formation and downstream PD readouts were detectable in mouse tissues (data not shown); therefore, mice could be used to preliminarily evaluate the therapeutic window for TAK-243. The dose-limiting toxicity observed in mice was a decrease in body weight. At the maximum tolerated

WSU-DLCL2
average tumor volume (mm3)
Table 2 TAK-243 tumor growth inhibition values in mice bearing xenograft tumors
Model Indication TGI
CWR22 Prostate 97
PHTX-235O Primary ovarian 97
HCC-70 Breast 95
PHTX-55B Primary breast 91
PHTX-24C Primary colon 87
HNM626 Primary neck 85
PHTX-132Lu Primary NSCLC 84
WSU-DLCL2 Lymphoma 83
HCT-116 Colon 83
Calu-6 NSCLC 52
MM1.S Multiple myeloma 80

Tumor-bearing mice were treated with TAK-243, and tumor growth inhibition (TGI) was calculated after the completion of dosing. As a standard protocol, mice were treated for 3 weeks with TAK-243 by intravenous administration at 2 doses/week (25 mg/kg/dose with the following exceptions: HCT-116 was dosed at 23 mg/kg, Calu-6 was dosed at 26 mg/kg, PHTX-235O received four doses (2 weeks dosing), HCC-70 received eight doses of 12.5 mg/kg for 4 weeks, PHTX-24C was dosed at 12.5 mg/kg, and PHTX-55B was dosed at 20 mg/kg). Each experiment included a vehicle control arm dosed with identical test article frequency. The percentage TGI was calculated within 5 d of the last dose, as described in the Online Methods. The TGI for the MM1.S model was calculated on day 15. The number of animals used/group was as follow: CWR (n = 10/group), PHTX-235O (n = 6/group), HCC-70 (n = 8/group), PHTX-55B (n = 5/group), PHTX-24C (n = 8/group), HNM626 (n = 10/group) and Calu-6 (n = 10/group). No animals were excluded from the analysis.

a c
Vehicle 25 mg/kg
18.75 mg/kg
12.5 mg/kg
2,000

1,500

1,000

500

0
0 5 10 15 20 25
Time (d)

1,500

Vehicle 25 mg/kg
18.75 mg/kg
12.5 mg/kg
PHTX-132Lu
average tumor volume (mm3)
1,000
dose in vivo (23–26 mg/kg), mean maximal body weight loss was
<12% of the animals’ body weight relative to that before the start of treatment (Supplementary Fig. 12b).
Two additional in vivo experiments were conducted to demonstrate that TAK-243 antitumor activity is driven through UAE inhibition. First, genetic knockdown of UAE expression, using a doxycycline (Dox)-inducible short hairpin RNA (shRNA) system, demonstrated comparable antitumor activity to that of TAK-243 in the HCT-116 xenograft model (Fig. 4b and Supplementary Fig. 13). In the sec- ond experiment, we used a xenograft mouse model with derivative of THP-1 cells (in which there is an Ala171Thr substitution in NAE that renders it resistant to pevonedistat) and demonstrated that TAK- 243 retained antitumor activity (Supplementary Fig. 14)28. Taken together, these data indicate that TAK-243 is UAE specific and has efficacy in a broad range of tumor models for both solid and hema- tological human cancers.

DISCUSSION
The diverse roles of ubiquitin in regulating cellular protein home- ostasis and ubiquitin signaling highlight the possibility of targeting the UPS to modulate human disease. Despite this potential, only a small fraction of the >500 enzymes involved in UPS have been tar- geted with agents that have entered human clinical studies. Here we report the identification of a first-in-class small-molecule inhibitor of UAE, TAK-243. TAK-243 is a potent inhibitor of UAE that results in complete inhibition of cellular ubiquitylation, leading to impaired ubiquitin-dependent proteolysis, ER stress, and impaired cell cycle progression and DNA damage repair. TAK-243 exposure caused robust loss of monoubiquitylation and polyubiquitylation of proteins and had substantial antitumor activity in preclinical models of human cancer. Previous small-molecule UAE inhibitors have demonstrated liabilities associated with much weaker potency and off-target activ- ity29,30. The data here validate TAK-243 as a new research tool for the acute modulation of cellular ubiquitylation activity both in vitro and
Figure 4 TAK-243 treatment has antitumor activity in mice bearing subcutaneous xenograft tumors. (a–d) TAK-243 antitumor activity, as assessed by tumor volume over time, was evaluated in mouse xenograft models bearing diffuse large B cell lymphoma (WSU-DLCL2) (a), colon cancer (HCT-116) (b), NSCLC (primary PHTX-132Lu model) (c) or multiple myeloma (MM1.S) (d) tumors. Mice with established tumors were dosed intravenously (starting on day 0) using either TAK-243 at the indicated doses or vehicle. Bortezomib (0.8 mg/kg) was used in the MM1. S model. Animals were treated biweekly on days 0, 3, 7, 10, 14 and 17. Data are mean tumor volume values (n = 10 mice per group); error bars represent the s.e.m. A two-tailed Welch’s t-test was used to compare antitumor activity between TAK-243-treated mice and vehicle-treated mice on day 21 (P < 0.001 for all models).

All of the mice were included in the analysisin vivo, and they support the assessment of TAK-243 in clinical trials in patients with advanced malignancies.Our cellular and in vivo data indicate that TAK-243 exerts its effects through UAE inhibition. Although TAK-243 inhibits UBA6 with equal potency in cells, UBA6 inhibition had no discernible effects on total ubiq- uitin conjugation or E2 thioester formation, with the expected excep- tion of the E2 enzyme USE1. Moreover, genetic knockout studies for UBA6 expression have failed to demonstrate significant effects on cancer cell viability31,32. In addition, any inhibition of NAE and the NEDD8- dependent cullin ring E3 ligases by TAK-243 would be superseded by the dominant role of UAE in supplying ubiquitin to all ligases. Consistent with this assertion, TAK-243 treatment did not result in the loss-of-func- tion phenotype characterized by DNA re-replication seen after inhibi- tion of NAE activity (as previously demonstrated with pevonedistat5). The findings that TAK-243 was efficacious in a model for NAE that was resistant to pevonedistat and was inactive when UAE expression was genetically knocked down support the conclusion that UAE inhibition is the mechanistic driver of TAK-243 antitumor activity.
Bortezomib, ixazomib and other compounds that target the fun- damental process of protein homeostasis demonstrate that a thera- peutic window can exist for patient benefit. Similarly, our preclinical data with TAK-243 suggest that a potential therapeutic window may exist for UAE inhibition. Mice tolerate TAK-243 with repeated dos- ing up to 25 mg/kg, doses that yield robust antitumor activity with minimal body weight loss or general organ toxicity. Additional comparative toxicological studies in rats and dogs (data not shown)
© 2018 Nature America, Inc., part of Springer Nature.

All rights reservedhave demonstrated that TAK-243 is safe to test in a human clinical trial (NCT02045095).
Loss of ubiquitylation activity, like proteasome inhibition, is expected to impair the degradation of short-lived proteins. Beyond its role in pro- teasome-related biology, additional functions of ubiquitin have become increasingly elucidated over the last several decades3,33. Receptor inter- nalization and lysosomal degradation, autophagy, signal transduction, transcription and DNA repair all involve ubiquitin modifications that are independent of K48-linked polyubiquitin chains. As a result, UAE inhibition, while sharing a number of mechanistic similarities with pro- teasome inhibition, also presents profound differences. For example, TAK-243 demonstrates substantial activity in a wide range of preclinical models of solid tumors, raising the hope that TAK-243 may demonstrate clinical benefit in solid tumor malignancies, whereas bortezomib and other proteasome inhibitors have yet to demonstrate consistent antitu- mor activity, both preclinically and clinically. For this reason, our pre- clinical investigation of TAK-243 focused mostly on solid tumors. It has been speculated that more profound disruption of protein homeostasis, above and beyond proteasome disruption, may be required for clinical activity in solid tumors; both TAK-243 and the recently disclosed VCP inhibitor CB-5083 (ref. 8), both upstream inhibitors of the ubiquitin proteasome system, represent new opportunities for the evaluation of this hypothesis. How differences between modulation of ubiquitin biol- ogy and proteasome inhibition might be exploited clinically is a matter of ongoing investigation. Notably, although we found that bortezomib and TAK-243 have similar EC50 values for inhibition of the cell viabil- ity of multiple-myeloma MM1.S cells in vitro, these compounds have marked differences in their antitumor efficacy in vivo when tested with MM1.S xenografts. Although these differences might be due to differ- ing PK properties of the compounds, they may also reflect the differing biological roles of their targets in the UPS pathway. The discovery of TAK-243 provides a valuable tool for the study of protein ubiquityla- tion and provides a new opportunity to study the modulation of protein homeostasis and ubiquitin signaling for the treatment of cancer.

METHODS
Methods, including statements of data availability and any associated accession codes and references, are available in the online version of the paper.
Note: Any Supplementary Information and Source Data files are available in the online version of the paper.

ACKNOWLEDGMENTS
The authors would like to thank W. Harper (Harvard Medical School) for the UBA6-knockout and control MEFs, A. Berger for critical review of the manuscript, and E. Koenig and P. Shah for genomic data analysis. We would also like to thank J. Afroze, I. Bharathan, J. Gaulin, M. Girad, C. McIntyre, F. Soucy, T.T. Wong and Y. Ye for performing the chemical synthesis of TAK-243. All activities were completed and funded through Takeda Pharmaceuticals Inc.

AUTHOR CONTRIBUTIONS
M.L.H., M.A.M., N.F.B., B.S.A., J.G. and L.R.D. participated in writing, reviewing and editing of the manuscript; M.L.H., M.A.M. and N.F.B. participated in the planning, initiation, data generation and analysis of biological experiments; J.C.,
S.L. and P.F. participated in the planning, initiation, design and execution of chemical synthesis; M.S. and N.B. performed crystallography experiments; J.P. was the toxicology representative on the program; J.G., T.S., F.B. and J.B. performed biochemical analyses; M.A.M., D.S., J.D., C.R., J.R. and K.H. performed in vitro cell culture experiments; M.L.H., T.T., J.H., J.S. and S.M.P. performed in vivo antitumor activity and pharmacodynamic experiments; A.L. and S.M. evaluated compound potencies in cell-based assays; Y.Y. and B.S. performed immunohistochemistry experiments; R.G. performed pharmacokinetic analyses; B.S.A., S.T., M.K., P.V., J.N., P.L., J.T.W., P.G., K.G., M.M. and C.C. provided project oversight and review.

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

Reprints and permissions information is available online at http://www.nature.com/ reprints/index.html. Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

1. Jin, J., Li, X., Gygi, S.P. & Harper, J.W. Dual E1 activation systems for ubiquitin differentially regulate E2 enzyme charging. Nature 447, 1135–1138 (2007).
2. Swatek, K.N. & Komander, D. Ubiquitin modifications. Cell Res. 26, 399–422 (2016).
3. Komander, D. & Rape, M. The ubiquitin code. Annu. Rev. Biochem. 81, 203–229 (2012).
4. Richardson, P.G. et al. A phase 2 study of bortezomib in relapsed, refractory myeloma. N. Engl. J. Med. 348, 2609–2617 (2003).
5. Brownell, J.E. et al. Substrate-assisted inhibition of ubiquitin-like protein-activating enzymes: the NEDD8 E1 inhibitor MLN4924 forms a NEDD8–AMP mimetic in situ. Mol. Cell 37, 102–111 (2010).
6. Huang, X. & Dixit, V.M. Drugging the undruggables: exploring the ubiquitin system for drug development. Cell Res. 26, 484–498 (2016).
7. Bedford, L., Lowe, J., Dick, L.R., Mayer, R.J. & Brownell, J.E. Ubiquitin-like protein conjugation and the ubiquitin–proteasome system as drug targets. Nat. Rev. Drug Discov. 10, 29–46 (2011).
8. Anderson, D.J. et al. Targeting the AAA ATPase p97 as an approach to treat cancer through disruption of protein homeostasis. Cancer Cell 28, 653–665 (2015).
9. Krönke, J. et al. Lenalidomide induces ubiquitination and degradation of CK1a in del(5q) MDS. Nature 523, 183–188 (2015).
10. McGrath, J.P., Jentsch, S. & Varshavsky, A. UBA1: an essential yeast gene encoding ubiquitin-activating enzyme. EMBO J. 10, 227–236 (1991).
11. Kulkarni, M. & Smith, H.E. E1 ubiquitin-activating enzyme UBA1 plays multiple roles throughout C. elegans development. PLoS Genet. 4, e1000131 (2008).
12. Misra, M. et al. Dissecting the specificity of adenosyl sulfamate inhibitors targeting the ubiquitin-activating enzyme. Structure 25, 1120–1129 (2017).
13. Amidon, B.S. et al. Indole-substituted pyrrolopyrimidinyl inhibitors of Uba6. US Patent 9593121B2. (2017).
14. Soucy, T.A. et al. An inhibitor of NEDD8-activating enzyme as a new approach to
treat cancer. Nature 458, 732–736 (2009).
15. Hershko, A. The ubiquitin system for protein degradation and some of its roles in the control of the cell division cycle. Cell Death Differ. 12, 1191–1197 (2005).
16. Gu, J.J. et al. MLN2238, a proteasome inhibitor, induces caspase-dependent cell death, cell cycle arrest, and potentiates the cytotoxic activity of chemotherapy agents in rituximab-chemotherapy-sensitive or rituximab-chemotherapy-resistant B cell lymphoma preclinical models. Anticancer Drugs 24, 1030–1038 (2013).
17. Milhollen, M.A. et al. Inhibition of NEDD8-activating enzyme induces re-replication and apoptosis in human tumor cells consistent with de-regulating CDT1 turnover. Cancer Res. 71, 3042–3051 (2011).
18. Ulrich, H.D. Ubiquitin and SUMO in DNA repair at a glance. J. Cell Sci. 125, 249–254 (2012).
19. Christianson, J.C. & Ye, Y. Cleaning up in the endoplasmic reticulum: ubiquitin in charge. Nat. Struct. Mol. Biol. 21, 325–335 (2014).
20. Moudry, P. et al. Ubiquitin-activating enzyme UBA1 is required for cellular response to DNA damage. Cell Cycle 11, 1573–1582 (2012).
21. Ungermannova, D. et al. Identification and mechanistic studies of a novel ubiquitin E1 inhibitor. J. Biomol. Screen. 17, 421–434 (2012).
22. Collins, A.R. The comet assay for DNA damage and repair: principles, applications and limitations. Mol. Biotechnol. 26, 249–261 (2004).
23. Vlachostergios, P.J., Patrikidou, A., Daliani, D.D. & Papandreou, C.N. The ubiquitin– proteasome system in cancer, a major player in DNA repair. Part 2: transcriptional regulation. J. Cell. Mol. Med. 13 9B, 3019–3031 (2009).
24. Wildey, G. et al. Pharmacogenomic approach to identify drug sensitivity in small- cell-lung cancer. PLoS One 9, e106784 (2014).
25. Wilmott, J.S. et al. BRAFV600E protein expression and outcome from BRAF inhibitor treatment in BRAFV600E metastatic melanoma. Br. J. Cancer 108, 924–931 (2013).
26. Barretina, J. et al. The Cancer Cell Line Encyclopedia enables predictive modeling of anticancer drug sensitivity. Nature 483, 603–607 (2012).
27. Huang, J. et al. NEDD8 inhibition overcomes CKS1B-induced drug resistance by upregulation of p21 in multiple myeloma. Clin. Cancer Res. 21, 5532–5542 (2015).
28. Milhollen, M.A. et al. Treatment-emergent mutations in NAEb confer resistance to the NEDD8-activating enzyme inhibitor MLN4924. Cancer Cell 21, 388–401 (2012).
29. Yang, Y. et al. Inhibitors of ubiquitin-activating enzyme (E1), a new class of potential cancer therapeutics. Cancer Res. 67, 9472–9481 (2007).
30. Xu, G.W. et al. The ubiquitin-activating enzyme E1 as a therapeutic target for the treatment of leukemia and multiple myeloma. Blood 115, 2251–2259 (2010).
31. Gilbert, L.A. et al. Genome-scale CRISPR-mediated control of gene repression and activation. Cell 159, 647–661 (2014).
32. Wang, T., Wei, J.J., Sabatini, D.M. & Lander, E.S. Genetic screens in human cells using the CRISPR–Cas9 system. Science 343, 80–84 (2014).
33. Yau, R. & Rape, M. The increasing complexity of the TAK-243 ubiquitin code. Nat. Cell Biol.
18, 579–586 (2016).

© 2018 Nature America, Inc., part of Springer Nature. All rights reserved.
ONLINE METHODS
Reagents. Pevonedistat, ixazomib, bortezomib and the UBA6 inhibitor (see compound I-01 in US patent 9593121 B2 (refs. 13,14) were synthesized at Takeda.