Research ArticleInflammation

CDK12-mediated transcriptional regulation of noncanonical NF-κB components is essential for signaling

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Science Signaling  31 Jul 2018:
Vol. 11, Issue 541, eaam8216
DOI: 10.1126/scisignal.aam8216

CDK12 promotes inflammation

Activating mutations in components of the noncanonical nuclear factor κB (NF-κB) signaling pathway are implicated in various cancers, and ligands that stimulate this pathway are increased in abundance in autoimmune inflammation, which makes this pathway an attractive therapeutic target. By combining a phenotypic screen with chemoproteomics analysis, Henry et al. found a compound that inhibited noncanonical NF-κB signaling by targeting cyclin-dependent kinase 12 (CDK12). By phosphorylating RNA polymerase II, CDK12 enables expression of the kinase NIK, which is required to stimulate the noncanonical NF-κB pathway. Together, these data suggest that CDK12 inhibitors may have therapeutic value in cancer and inflammatory disease.

Abstract

Members of the family of nuclear factor κB (NF-κB) transcription factors are critical for multiple cellular processes, including regulating innate and adaptive immune responses, cell proliferation, and cell survival. Canonical NF-κB complexes are retained in the cytoplasm by the inhibitory protein IκBα, whereas noncanonical NF-κB complexes are retained by p100. Although activation of canonical NF-κB signaling through the IκBα kinase complex is well studied, few regulators of the NF-κB–inducing kinase (NIK)–dependent processing of noncanonical p100 to p52 and the subsequent nuclear translocation of p52 have been identified. We discovered a role for cyclin-dependent kinase 12 (CDK12) in transcriptionally regulating the noncanonical NF-κB pathway. High-content phenotypic screening identified the compound 919278 as a specific inhibitor of the lymphotoxin β receptor (LTβR), and tumor necrosis factor (TNF) receptor superfamily member 12A (FN14)–dependent nuclear translocation of p52, but not of the TNF-α receptor–mediated nuclear translocation of p65. Chemoproteomics identified CDK12 as the target of 919278. CDK12 inhibition by 919278, the CDK inhibitor THZ1, or siRNA-mediated knockdown resulted in similar global transcriptional changes and prevented the LTβR- and FN14-dependent expression of MAP3K14 (which encodes NIK) as well as NIK accumulation by reducing phosphorylation of the carboxyl-terminal domain of RNA polymerase II. By coupling a phenotypic screen with chemoproteomics, we identified a pathway for the activation of the noncanonical NF-κB pathway that could serve as a therapeutic target in autoimmunity and cancer.

INTRODUCTION

The noncanonical nuclear factor κB (NF-κB) pathway plays a critical role in the development and homeostatic control of the immune system. Mutations that activate noncanonical NF-κB are observed in some cancers, including multiple myeloma, lymphoma, and leukemia (1). Ligands that activate noncanonical NF-κB signaling, such as B cell–activating factor (BAFF), lymphotoxin α1β2 heterotrimer, tumor necrosis factor (TNF)–related weak inducer of apoptosis (TWEAK), receptor activator of NF-κB ligand (RANKL), and OX40 antigen ligand, are increased in abundance in human autoimmune diseases, including systemic lupus erythematosus, Sjögren’s syndrome, scleroderma, rheumatoid arthritis, inflammatory bowel disease, and multiple sclerosis (2). Thus, inhibitors of noncanonical NF-κB signaling could potentially ameliorate a wide range of inflammatory diseases, cancer, and fibrosis.

The accumulation of NF-κB–inducing kinase (NIK, which is encoded by MAP3K14) and the processing of p100 (encoded by NFKB2) to p52 are two hallmarks of noncanonical NF-κB signaling and differentiate the noncanonical from the canonical NF-κB signaling pathway. In the basal state, a ubiquitin ligase complex composed of TNF-associated factor 2 (TRAF2), TRAF3, and cellular inhibitor of apoptosis proteins (cIAPs), constitutively induces the degradation of NIK (3, 4). Ligand binding to certain TNF family receptors (TNFRs) disrupts the interaction of NIK with this ubiquitin ligase complex. As a result, the ubiquitin ligase complex is degraded, enabling NIK to accumulate because of new protein synthesis (5). NIK phosphorylates NF-κB inhibitor κB (IκB) kinase α (IKKα), which in turn phosphorylates p100 and induces the proteasomal-dependent cleavage of p100 to generate p52 (6). As a consequence of this p100 processing, p52-containing transcription factor complexes translocate to the nucleus. Thus, noncanonical NF-κB signaling requires NIK accumulation to induce the processing of p100 to p52. In contrast, canonical NF-κB complexes composed of Rel homodimers or Rel-p50 heterodimers are held in the cytoplasm by inhibitory IκB proteins. Upon TNFR ligand binding, a complex of IKKα, IKKβ, and IKKγ oligomerizes on scaffolds of linear or Lys63 (K63)–linked ubiquitin chains. These oligomerization events activate IKKβ, which phosphorylates and targets IκB proteins for proteasomal degradation, thereby enabling canonical NF-κB transcription factors to translocate to the nucleus.

To identify small molecules that selectively inhibited the noncanonical NF-κB pathway and spared the canonical NF-κB pathway, we performed a high-content phenotypic screen in U-2 OS cells, a human osteosarcoma cell line. This screen identified compounds that inhibited the nuclear translocation of p52 stimulated by either an agonistic antibody against the lymphotoxin β receptor (anti-LTβR) or TWEAK but did not inhibit the TNF-α–mediated nuclear translocation of p65 (also known as RelA). Using a chemoproteomics approach (79) to identify the molecular mechanism of action of a lead compound, we discovered that the cyclin-dependent kinase 12/cyclin K complex (CDK12/CCNK) promoted the ligand-induced increase in the abundances of MAP3K14 and NFKB2 mRNAs. By inhibiting CDK12/CCNK, the lead compound 919278 prevented the accumulation of NIK, thus impairing activation of the noncanonical NF-κB pathway. These findings elucidate a previously unknown aspect of noncanonical NF-κB pathway regulation.

RESULTS

Compound 919278 selectively inhibits the noncanonical NF-κB pathway

A high-throughput screen (HTS) for inhibitors of the noncanonical NF-κB pathway identified several structurally different chemical scaffolds, of which 919278 is the focus of this report. Binding of an agonistic antibody to LTβR (anti-LTβR) or binding of TWEAK to the receptor FN14 induced p52 nuclear translocation in U-2 OS cells (Fig. 1A), whereas TNF-α binding to TNFR predominantly activated the canonical NF-κB pathway, leading to p65 nuclear translocation (Fig. 1A). We validated the use of monitoring p52 and p65 nuclear translocation as effective assays of NIK-dependent noncanonical NF-κB activity and IKKβ-dependent canonical NF-κB activity, respectively, using the NIK inhibitor Amgen16 (Fig. 1A) (10) and the IKKβ inhibitor ACHP (2-amino-6-[2-(cyclopropylmethoxy)-6-hydroxyphenyl]-4-(4-piperidinyl)-3 pyridinecarbonitrile) (fig. S1) (11). The assay with ACHP confirmed that a 30-min pretreatment with the inhibitor followed by a 30-min stimulation with TNF-α was an appropriate paradigm to monitor canonical pathway activation and inhibition (fig. S1). Similar to Amgen16, a 30-min pretreatment with compound 919278 inhibited p52 nuclear translocation in response to a 4-hour stimulation with anti-LTβR [median inhibitory concentration (IC50) = 0.169 μM] or with TWEAK (IC50 = 0.167 μM) and did not inhibit p65 nuclear translocation (Fig. 1B). Thus, 919278 regulated the noncanonical pathway in a ligand-independent manner and selectively inhibited the noncanonical pathway while sparing the canonical NF-κB signaling pathway. Compound 919278 contains a stereogenic center and is the (R) enantiomer. To explore the importance of the stereochemistry of 919278, we tested the (S) enantiomer compound 702697 (Fig. 1, B and C). The enantiomer 702697 (IC50 ≈ 10 μM) was much less potent than 919278 in both the anti-LTβR– and TWEAK-stimulated p52 translocation assays (702697 IC50 ≈ 10 μM, 919278 IC50 ≈ 0.17 μM) and did not alter TNF-α–induced p65 nuclear translocation (Table 1). Thus, the stereochemistry of these molecules is important, and 702697 served as a negative control for the mechanistic study of 919278.

Fig. 1 p52 nuclear translocation in U-2 OS cells is inhibited by the compound 919278.

(A) Top and middle: p52 nuclear translocation in U-2 OS cells was determined by measuring the ratio of nuclear to cytoplasmic staining of p52/p100 protein. Cells were pretreated with 7.5 μM Amgen16, 1 μM 919278, or 1 μM 702697 for 30 min and then were exposed to the agonistic antibody to LTβR (anti-LTβR, 20 ng/ml; top) or TWEAK (20 ng/ml; middle) for 4 hours. Unstimulated cells were exposed to dimethyl sulfoxide (DMSO). Bottom: p65 nuclear translocation in U-2 OS cells was determined by measuring the ratio of nuclear to cytoplasmic staining of p65 protein. Cells were pretreated with 7.5 μM Amgen16, 1 μM 919278, or 1 μM 702697 for 30 min and then exposed to TNF-α (10 ng/ml) for 30 min. Unstimulated cells were exposed to DMSO. Images are representative of three independent experiments. Right: Structures of the compounds used. (B) Percentage inhibition of the nuclear translocation of the indicated transcription factors from cells treated as described in (A) using a fourfold serial dilution of the indicated inhibitors. The stimulants used and transcription factors measured are indicated above the graphs. Data are means ± SD of three independent experiments. (C) Compound structures.

Table 1 IC50 values.

The IC50 values (μM) are for compounds tested in U-2 OS cells by nuclear translocation assay for p52 by TWEAK or anti-LTβR and for p65 by TNF-α.

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Chemoproteomics identifies CDK12 and CCNK as targets of 919278

To determine the mechanism of action of 919278, we sought to identify its target(s). Because the noncanonical NF-κB signaling pathway involves several phosphorylation events, we first interrogated kinases as a potential class of proteins that 919278 might inhibit. We tested a racemic mixture of 919278 and 702697 at a final concentration of 1 μM in a Reaction Biology Corporation enzymatic assay panel (table S1), in the DiscoverX KINOMEscan binding assay (table S2), and at a final concentration of 10 μM in the ActivX KiNativ assay (table S3). None of these panels identified putative targets for 919278.

Lacking any identified targets using commercially available panels, we used an in-house chemoproteomics approach to profile the U-2 OS cell kinome. Chemoproteomics is a mass spectrometry (MS)–based method that uses small-molecule probes to enrich and identify protein complexes to which a drug candidate binds (7). For example, under near physiological conditions, the selectivity of kinase inhibitors can be determined by assessing their ability to compete for kinome binding to promiscuous kinase inhibitors immobilized on affinity beads (Fig. 2A). When a small molecule binds to its target kinase, the abundance of that kinase bound to the affinity beads is reduced in a dose-dependent manner. Kinases that do not bind to the small molecule and that are not in a complex with a kinase that binds to the small molecule will maintain similar abundances in the captured subproteomes across all small-molecule concentrations. The captured subproteome is eluted and digested with trypsin, which is followed by sequential coupled liquid chromatography tandem MS (LC-MS/MS) quantitation.

Fig. 2 Chemoproteomics profiling through competition measurements identifies CDK12 and CCNK as interaction partners of compound 919278.

(A) Flowchart describing the chemoproteomics approach used to identify unknown target(s) of a compound. Briefly, cells containing endogenous amounts of protein are incubated with the drug or control. Cells are lysed, and beads conjugated to nonselective kinase inhibitors are added to the drug-lysate mixture. The inhibitor-conjugated beads compete with the drug for binding to the target. Binding of a drug to its target reduces the amount of target protein in the enriched subproteome, which is detected by LC-MS/MS. Diagram modified from Bantscheff et al. (7). (B) Effect of 919278 on CDK12 and CCNK recovery from U-2 OS cells. Untreated (DMSO) or TWEAK-stimulated U-2 OS cells were incubated with increasing concentrations of 919278, and the amounts of CDK12 (left) and CCNK (right) recovered after incubation with inhibitor-conjugated beads were determined. Data are means ± SD of three technical replicates from a single experiment and are representative of three independent experiments. (C) Effect of knocking down CDK12 on anti-LTβR–stimulated p52 nuclear localization. U-2 OS cells were transfected with nontargeting (NT) siRNA or with the CDK12-specific siRNA s525626. Three days later, the cells were evaluated for p52 nuclear translocation after 4 hours under unstimulated or stimulated (anti-LTβR antibody) conditions. Images are representative of three independent experiments. (D) Percentage of responding U-2 OS cells that were transfected with nontargeting siRNA and then treated with or without (Unstim.) anti-LTβR antibody or transfected with CDK12-specific siRNA and then stimulated with anti-LTβR antibody. Data are means ± SEM of three independent experiments. **P = 0.0011, ***P = 0.0008, ****P < 0.0001 by one-way analysis of variance (ANOVA) and Tukey’s multiple comparisons test. (E) Structure of THZ1. (F) Percentage inhibition of TWEAK-induced p52 nuclear translocation (calculated from the ratio of nuclear to cytoplasmic staining of p52/p100), using a fourfold serial dilution of THZ1. Data are means ± SD of three independent experiments.

We applied chemoproteomics to ~2200 proteins from U-2 OS cells, of which ~250 were kinases (table S4). Using a competitive, kinase-targeted chemoproteomics approach that included four different nonselective kinase inhibitors (Fig. 2A and fig. S2), we identified the CDK12/CCNK complex as a potential target of 919278. Compound 919278 reduced the binding of both CDK12 and its associated protein, CCNK, to the kinase affinity beads in TWEAK-stimulated and unstimulated U-2 OS cells compared to DMSO controls, with IC50 values ranging from 50 to 61 nM for CDK12 and from 29 to 68 nM for CCNK (Fig. 2B).

We took both genetic and pharmacological approaches to verify that the binding of 919278 to CDK12 inhibited noncanonical NF-κB signaling. We knocked down CDK12 in U-2 OS cells with small interfering RNA (siRNA) and observed a reduction in p52 nuclear translocation in response to anti-LTβR (Fig. 2C). The average reduction in CDK12 mRNA abundance by multiple siRNAs was 76%, and we observed a 49% decrease in cells responding to anti-LTβR with p52 nuclear translocation (Fig. 2D and Table 2). Similarly, THZ1 (Fig. 2E), an inhibitor of both CDK7 and CDK12 (12), inhibited TWEAK-induced p52 nuclear translocation with an IC50 value of 0.015 μM (Fig. 2F). Knockdown of CCNK had no effect on p52 translocation (table S5). We then tested 919278 and 702697 for their ability to bind to CDK12 in a DiscoverX CDK12-binding assay that was not part of the kinome panel that we initially tested, which revealed a Kd (dissociation constant) of 5.6 μM for 919278 and no detectable activity for 702697 at concentrations up to 30 μM (Table 3).

Table 2 Percentage inhibition of anti-LTβR–induced p52 nuclear translocation in U-2 OS cells treated with siRNAs.

KD, knockdown; ND, not determined.

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Table 3 Binding affinities for compounds in the DiscoverX CDK12 assay.
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The 919278 enantiomer has a more favorable interaction in a CDK12 adenosine 5′-triphosphate–binding site model than does 702697

To visualize the binding of 919278 to CDK12, we used molecular dynamics simulations to dock 919278 and 702697 into the adenosine 5′-triphosphate (ATP)–binding site of CDK12. Docking of 919278 and 702697 to this site [using the crystal structure in Protein Data Bank (PDB): 4NST] did not produce binding poses with two hinge interactions. Therefore, we modeled the two inhibitors manually based on the binding modes of analogous inhibitors found in the crystal structures of two different kinases (PDB codes 4FOC and 4BHN). The two inhibitors were modeled to mimic the two hinge-binding interactions and minimized using an MOE modeling program. The 4FOC-like hinge H-bonding conformation yielded the lowest interaction energy (−9.2 kcal/mol) for 919278 (fig. S3) and a slightly higher interaction energy (−8.3 kcal/mol) for 702697. In the 4BHN-like hinge H-bonding conformations, the ligands had a greater interaction energy of about 1 kcal/mol, respectively.

919278 inhibits the cellular activity of CDK12

CDK12 activates RNA polymerase (Pol) II–mediated transcription by phosphorylating serine-2 (Ser2) within the 52 heptad (Y1S2P3T4S5P6S7) repeats in the C-terminal domain (CTD) of RNA Pol II (13, 14). These Ser2 phosphorylation events aid in the release of paused RNA Pol II from promoters, resulting in transcriptional elongation, which is particularly important for the transcription of some long, complex genes. We therefore evaluated whether 919278, 702697, or THZ1 changed the extent of site-specific phosphorylation in the CTD of RNA Pol II. Both 919278 and THZ1 reduced the phosphorylation of Ser2 of the RNA Pol II CTD (Fig. 3 and fig. S4). THZ1 also substantially reduced the phosphorylation of Ser5 and total RNA Pol II, whereas 919278 did not (Fig. 3 and fig. S4). The effect of 919278 on the phosphorylation of Ser2 and not Ser5 is consistent with a CDK12 inhibitor that selectively reduces the phosphorylation of Ser2 in RNA Pol II, without affecting Ser5 phosphorylation (15). As expected, 702697 did not substantially affect the phosphorylation of either Ser2 or Ser5 (Fig. 3 and fig. S4). Together, these data suggest that 919278 inhibits the kinase activity of CDK12.

Fig. 3 919278 inhibits CDK12 cellular activity and reduces the phosphorylation of Ser2 on the RNA Pol II CTD.

Western blotting analysis of the phosphorylation of the RNA Pol II CTD. U-2 OS cells were pretreated for 30 min with the indicated compounds and then left unstimulated (−) or stimulated with TWEAK (20 ng/ml; +) for 4 hours. The amounts of RNA Pol II CTD phosphorylated at Ser2 and Ser5 were normalized to total RNA Pol II CTD, which was itself normalized to the amount of β-actin and are indicated by the numbers under the blots. For quantification of three independent experiments, see fig. S4.

Knockdown of CDK12 phenocopies 919278-induced transcriptome changes

To validate 919278 as a CDK12 inhibitor, we compared the effect of knockdown of CDK12 with that of treatment with 919278 on gene expression in either unstimulated or TWEAK-stimulated U-2 OS cells (Fig. 4 and fig. S5). Controls included DMSO (vehicle), 702697, and the NIK inhibitor Amgen16. Cluster analysis of Spearman correlation coefficients [the rank order of differentially expressed genes (DEGs) in tables S6 and S7] showed that the effects of CDK12 knockdown by two individual siRNAs at 4 and 24 hours were similar to the effects of 919278 at 24 hours (Fig. 4, red boxes) in both unstimulated and TWEAK-stimulated U-2 OS cells. In contrast, Amgen16, 702697, DMSO, and the nontargeting control siRNAs correlated less well with compound treatment (Fig. 4, blue boxes). We also determined Spearman correlation coefficients for DEGs in response to TWEAK for each condition (fig. S5), and we analyzed those genes that were differentially expressed in response to TWEAK and the effects of the various treatments on their expression (table S6). These data support the conclusion that 919278 targets CDK12 and showed that CDK12 inhibition with a small molecule or knockdown with siRNA resulted in similar profiles of DEGs.

Fig. 4 RNA-seq reveals that 919278 phenocopies the effect of CDK12 knockdown on DEGs.

Similarity of transcription profiles (of the genes differentially expressed in response to TWEAK stimulation) in response to 919278 treatment (24 hours) compared with the transcription profiles in response to the indicated siRNAs or compounds. Comparison was performed by hierarchical clustering of Spearman rank coefficients (rank-order similarity) using the 505 DEGs from U-2 OS cells. Cells were treated with CDK12-specific siRNA or nontargeting siRNA for 72 hours or were pretreated for 30 min with 919278 and 702697 (each at 1 μM), Amgen16 (7 μM), or DMSO and then left unstimulated (Unstim) or stimulated with TWEAK (20 ng/ml) for 24 hours. Red indicates sample profiles that are similar; blue indicates sample profiles that are more distant. Data are from two biological replicates and are a subset of those shown in fig. S5.

CDK12 inhibitors block the TWEAK-induced increase in MAP3K14 and NFKB2 mRNA abundances

Our transcript-profiling data (table S6) indicated that TWEAK induced increases in MAP3K14 and NFKB2 mRNA abundance in a CDK12-dependent manner, suggesting that CDK12 functions in the noncanonical NF-κB pathway. To confirm these findings, we measured the abundances of MAP3K14 (which encodes NIK) and NFKB2 (which encodes p100) mRNAs in U-2 OS cells in response to TWEAK in the presence or absence of various compounds. We found that TWEAK increased MAP3K14 and NFKB2 mRNA abundance by 2.3- and 4.5-fold, respectively (Fig. 5A). In contrast, TWEAK did not alter the mRNA abundances of either the CDK12-regulated gene RAD51 (16) or CDK12 itself. The degree of reduction in CDK12 mRNA abundance by siRNA-mediated knockdown correlated with the degree to which MAP3K14 and NFKB2 mRNA abundances were decreased (R2 = 0.79 and R2 = 0.86, respectively) in cells stimulated with the anti-LTβR antibody (Fig. 5B), indicating that CDK12 is required for the stimulated expression of genes encoding components of the noncanonical NF-κB pathway. Both 919278 and THZ1 reduced MAP3K14 mRNA abundance (Fig. 5C) with IC50 values of 0.32 ± 0.053 μM and 0.032 ± 0.022 nM, respectively (fig. S6A). We found that 919278 was ≥26 times more potent than 702697 in its ability to reduce MAP3K14 mRNA abundance (919278 IC50 = 0.32 ± 0.053 μM, 702697 IC50 ≥ 8.4 μM; Fig. 5C and fig. S6A). Similarly, 919278, but not 702697, reduced the abundance of NFKB2 (Fig. 5D and fig. S6A), CDK12, and RAD51 (fig. S6A) mRNAs. Reductions in MAP3K14 and NFKB2 mRNA abundance by 919278 and THZ1 were similar in both unstimulated and TWEAK-stimulated cells (Fig. 5, C and D) and were consistent with the effects of CDK12 knockdown (Fig. 5B). Together, these data support the interpretation that MAP3K14 and NFKB2 mRNA amounts are dynamic, highly regulated, and influenced by CDK12 activity.

Fig. 5 Compound 919278 inhibits ligand-mediated induction of the noncanonical NF-κB pathway mediator-encoding genes MAP3K14 and NFKB2.

(A) Relative abundances of MAP3K14, NFKB2, CDK12, and RAD51 mRNAs in U-2 OS cells after 4 hours of incubation without TWEAK (− TWEAK) or with TWEAK (20 ng/ml; + TWEAK). Data were normalized to the mRNA amount in the absence of TWEAK and are plotted as means ± SEM of three independent experiments. ****P < 0.0005 compared to untreated (− TWEAK) by two-way ANOVA with Sidak’s multiple comparisons test. (B) Correlation between CDK12 knockdown efficiency and reduction in MAP3K14 or NFKB2 mRNA abundance. The abundances of CDK12, MAPK3K14, and NFKB2 mRNAs were determined for U-2 OS cells on day 3 after transfection with CDK12-specific siRNA. R2 = 0.79 (MAP3K14) and 0.86 (NFKB2). (C and D) Relative abundances of MAP3K14 (C) and NFKB2 (D) mRNAs in U-2 OS cells pretreated for 30 min with DMSO (vehicle), 1 μM Amgen16, 1 μM 919278, 1 μM 702697, or 0.5 μM THZ1 and then left unstimulated or stimulated for 4 hours with TWEAK, TNF-α, or anti-LTβR antibody, as indicated. Data are representative of three independent experiments. *P < 0.05, **P < 0.005, ****P < 0.0005 by two-way ANOVA and Dunnett’s multiple comparisons test. (E) Relative abundance of MAP3K14 mRNA in primary human peripheral B cells pretreated for 30 min with DMSO, 10 μM 919278, 10 μM 702697, 1 μM THZ1, or 10 μM Amgen16 and then left unstimulated or stimulated for 4 hours with CD40L (500 ng/ml). Data are means ± SEM of three experiments with cells from different donors. **P < 0.005 and ***P < 0.0001 compared to the CD40L-stimulated, DMSO-pretreated control. Data were analyzed by one-way ANOVA and Dunnett’s multiple comparisons test.

NFKB2 transcription is induced in response to ligands that stimulate canonical NF-κB signaling (17); however, two ligands that stimulate noncanonical NF-κB signaling (TWEAK and RANKL) also induce NFKB2 transcription (18, 19). Consistent with these reports, we observed an increase in NFKB2 mRNA abundance with either noncanonical (TWEAK or anti-LTβR antibody) or canonical NF-κB stimuli (TNF-α; Fig. 5D). Whereas TWEAK- and anti-LTβR–mediated induction of NFKB2 transcription was almost absent in the presence of the NIK inhibitor Amgen16, TNFR-mediated NFKB2 transcription was statistically significantly decreased, but still induced, in the presence of the NIK inhibitor (Fig. 5D). These data suggest that both the noncanonical and canonical NF-κB transcription factors stimulate NFKB2 expression and that only those stimuli that activate the noncanonical pathway depend on NIK. In contrast, we observed increased MAP3K14 mRNA abundance in response to TWEAK and anti-LTβR but not in response to TNF-α (Fig. 5C). Moreover, Amgen16 had little effect on the abundance of MAP3K14 mRNA (Fig. 5C). Thus, the regulation of NFKB2 and MAP3K14 expression appeared to involve different mechanisms. Both MAP3K14 and NFKB2 transcripts were reduced in abundance in the presence of 919278 and THZ1, regardless of the stimulus (Fig. 5, C and D), indicating that both genes depended on CDK12 activity for induction. Together, these data suggest that inhibition of CDK12 activity results in reduced basal- and stimulation-induced transcription of MAP3K14 and NFKB2.

To address whether this paradigm holds in other cell types, we measured the effect of CDK12 inhibition on the response to CD40L in primary human B lymphocytes (Fig. 5E). CD40 engagement activates both the canonical and noncanonical NF-κB pathways to support B cell proliferation after a productive interaction with activated T cells (20). There was a slight, but not statistically significant (P = 0.26), increase in MAP3K14 mRNA abundance in primary human B cells after stimulation with CD40L. Exposing the cells to 10 μM 919278 or 1 μM THZ1 decreased the MAP3K14 mRNA abundance below that of the unstimulated controls. In contrast, 10 μM 702697 or Amgen16 did not inhibit, and even slightly enhanced, the abundance of MAP3K14 mRNA (Fig. 5E). With the exception of the limited induction in response to stimulating ligand, these data are consistent with the MAP3K14 expression in U-2 OS cells stimulated with TWEAK or the anti-LTβR antibody in the presence of the CDK12 inhibitor. The effect of 919278 on NFKB2 transcripts in B cells (fig. S6B) paralleled that of 919278 on MAP3K14 mRNA abundance. There was a slight increase (P = 0.10) in NFKB2 transcript abundance in primary human B cells after stimulation with CD40L. We found that 919278 reduced NFKB2 transcript abundance in two of three donors; however, three of three donors showed a decrease in MAP3K14 mRNA abundance. Because NFKB2 expression is strongly induced by canonical NF-κB activation and 919278 does not inhibit canonical complexes well, it is possible that this donor may have had more activated B cells. Together, these results suggest that CD40L increases MAP3K14 and NFKB2 transcript abundance in peripheral B cells and that the expression of these genes depends on CDK12 and is reduced in the presence of the inhibitors THZ1 and 919278.

Compound 919278 inhibits NIK accumulation in TWEAK-stimulated cells

To determine whether 919278 affected other components of the canonical or noncanonical NF-κB pathways, we examined the effect of the compounds in the abundance of the TWEAK receptor FN14 at the cell surface, cell viability, and the abundance of cIAP1 and NIK. We assessed the abundance of FN14 at the cell surface by flow cytometry (fig. S7A). TWEAK treatment reduced the cell surface abundance of FN14, consistent with TWEAK-induced FN14 internalization (Fig. 6A and fig. S7B). However, Amgen16, 919278, and THZ1 did not alter cell surface FN14 abundance in unstimulated or TWEAK-stimulated cells. Reduced FN14 surface abundance occurred to an equal extent in both compound- and DMSO-treated cells (Fig. 6A and fig. S7B). These data support the hypothesis that CDK12 is downstream of receptor engagement.

Fig. 6 Compound 919278 acts upstream of NIK accumulation in TWEAK-stimulated U-2 OS cells.

(A) Abundance of FN14 at the surface of U-2 OS cells. Cells were pretreated for 30 min with indicated compounds and then were left unstimulated (− TWEAK) or were stimulated for 30 min with TWEAK (20 ng/ml; + TWEAK). Cell surface FN14 abundance is shown as median fluorescence intensity (MFI) as determined by flow cytometry (see fig. S7 for gating strategy and representative histograms). Data are means ± SEM of three independent experiments normalized to unstimulated DMSO controls. There were no statistically significant differences between compound treatments and DMSO. TWEAK induced a statistically significant reduction in surface abundance of FN14 regardless of compound treatment. P ≤ 0.007 by two-way ANOVA and Sidak’s multiple comparisons test. (B) Western blotting (IB) analysis of the relative abundances of cIAP1, NIK, p100, and p52 in U-2 OS cells pretreated for 30 min with the indicated compounds and then left unstimulated (−) or stimulated with TWEAK (20 ng/ml; +) for 4 hours. β-Actin served as the loading control. See fig. S7D for quantification of the ratio of p52 to p100 abundance (p52/p100) from three independent experiments. (C) Western blotting analysis of the relative abundance of NIK in U-2 OS cells pretreated for 30 min with the indicated compounds and then stimulated with TWEAK for 4 hours. β-Actin served as the loading control. Images are representative of two independent experiments.

We next evaluated the effect of compound treatment on cell viability by determining the number of cell nuclei per well. The number of nuclei per well in each condition was normalized to a DMSO control and compared to the inactive enantiomer 702697. The viability of cells treated with various concentrations of 919278, THZ1, or Amgen16 was not statistically significantly different from that of U-2 OS cells treated with 702697 (fig. S7C). Therefore, cell viability was not a confounding factor in these experiments.

To assess differences in stimulation-dependent degradation of cIAP1, we monitored cIAP1 abundance by Western blotting analysis of unstimulated and TWEAK-stimulated cells in the presence or absence of the compounds. We found that TWEAK induced the degradation of cIAP1, regardless of compound treatment, indicating that CDK12 inhibition did not impair the TWEAK-mediated inactivation of the cIAP-TRAF3 ubiquitin ligase that targets NIK for proteasomal degradation (Fig. 6B). Thus, the effect of CDK12 on noncanonical NF-κB signaling is downstream of this cIAP-containing ubiquitin ligase complex, which could lead to the stabilization of NIK. Thus, we examined NIK abundance and p100 processing. After cIAP is degraded, NIK protein accumulates, phosphorylates IKKα, and causes p100 cleavage to p52 (5). Exposing cells to 919278 blocked TWEAK-induced NIK accumulation in a concentration-dependent manner (Fig. 6C). THZ1 also prevented TWEAK-induced NIK accumulation, whereas neither 702697 nor the NIK inhibitor Amgen16 affected NIK protein abundance (Fig. 6B). As expected, 919278, THZ1, and Amgen16 blocked the processing of p100 to p52 (Fig. 6B and fig. S7D). In contrast, the enantiomer 702697 did not reduce p100 processing. Because NIK is required for the noncanonical NF-κB pathway, these data suggest that 919278 predominantly inhibits noncanonical NF-κB signaling by impairing MAP3K14 expression through CDK12 inhibition and not by preventing ligand-induced stabilization of NIK. Consistent with the model of regulation of noncanonical NF-κB signaling at the level of mRNA, we observed that the transcriptional inhibitor actinomycin D phenocopied 919278: Both compounds blocked NIK accumulation and inhibited the processing of p100 to p52 (Fig. 6B). Together, our data suggest that 919278 regulates MAP3K14 expression at the transcriptional level, rather than at the level of mRNA translation or NIK protein degradation.

DISCUSSION

Here, we identified CDK12-regulated transcription of MAP3K14 and NFKB2 as a previously uncharacterized mechanism for the regulation of the noncanonical NF-κB pathway. The noncanonical NF-κB pathway is an attractive target for the treatment of some autoimmune diseases and cancers. The successful use of therapeutic antibodies, such as anti-BAFF (Belimumab), to inhibit specific receptors that are part of the noncanonical pathway suggests that targeting individual noncanonical ligands or receptors can ameliorate disease symptoms (21). It stands to reason that targeting a central node of the noncanonical NF-κB pathway, such as NIK, could provide broader efficacy. However, despite drug development efforts, there is no approved treatment to inhibit pan noncanonical NF-κB signaling, NIK activation, or both. Moreover, contrary to the previous dogma that NIK protein abundance is primarily regulated at the posttranslational level, our data highlight a previously underappreciated regulation of NIK at the level of gene transcription. Our observation that pathway stimulation can increase MAP3K14 mRNA abundance is consistent with that of another report (19). Although it is well established that NFKB2 mRNA abundance increases with canonical NF-κB stimulation (22), our results demonstrate that CDK12 is a key kinase involved in transcriptional control of both MAP3K14 and NFKB2 mRNA abundance under basal conditions and in response to stimuli of noncanonical NF-κB signaling. CDK12 was previously identified as a potential target for cancer treatment (15), and our data suggest that CDK12 could broadly inhibit noncanonical NF-κB signaling. Thus, CDK12 could be a particularly attractive target in the treatment of cancers, such as multiple myeloma, in which noncanonical NF-κB signaling is increased (1). The role of CDK12 in transcriptional regulation of the noncanonical NF-κB pathway highlights the need for more research of this pathway. Clarity on how other kinases and cell processes feed into the noncanonical NF-κB pathway may elucidate other druggable targets.

Our results also support the use of phenotypic screens in drug development efforts. Our high-content phenotypic screen in U-2 OS cells was designed around the characteristic nuclear translocation of the Rel-p52 complex in response to activation of the noncanonical NF-κB pathway. This phenotypic screening method led to the nonbiased identification of 919278 as a compound that could selectively inhibit noncanonical NF-κB signaling but spare canonical NF-κB signaling. The inability of 919278 to inhibit the TNF-α–induced nuclear translocation of p65 provided the initial evidence suggesting its specificity for the noncanonical pathway. Moreover, the 20- to 30-fold reduced potency of the (S) enantiomer 702697 in the p52 nuclear translocation assay supplied us with a negative control for deconvolution efforts. We were able to successfully identify a functional target of 919278 using a chemoproteomics assay conducted on the same cells used in the screening assay, which illustrates the importance of physiologic and cellular context in target identification strategies. None of the other kinase panel profiling approaches led to the revelation that 919278 inhibits CDK12. Note that 919278, but not 702697, depleted CDK12 and CCNK from the subproteome, suggesting that 919278 interacts with one or both of these proteins. Further validation of CDK12 with CDK12-targeting siRNA confirmed the role of CDK12 in noncanonical NF-κB signaling. Note that CCNK-specific siRNA had no such effect. The lack of effect of CCNK-specific siRNA on p52 nuclear translocation may be due to insufficient knockdown of CCNK or to another factor compensating for the loss of CCNK. CCNK was likely detected in the chemoproteomics study because of its association with CDK12 rather than a direct interaction with 919278, because the chemoproteomics assay was directed at the ATP-binding sites of kinases, and CCNK is not a kinase. Hence, CCNK likely is not a direct target of 919278 and may not be necessary or sufficient for transcriptional regulation of MAP3K14 and NFKB2. Thus, it is possible that other cyclins can replace CCNK as a cofactor for CDK12. For these reasons, we focused on CDK12 as the primary target of 919278.

We conducted small-molecule and siRNA screens with two different stimuli (an anti-LTβR agonist antibody and TWEAK) to ensure that the pathway targets of interest were central to general noncanonical NF-κB signaling rather than specific to one particular stimulus. Using both directed quantitative polymerase chain reaction (qPCR) and RNA sequencing (RNA-seq) analyses, we found that 919278 reduced the abundances of MAP3K14 and NFKB2 mRNAs, which encode two key components of noncanonical NF-κB signaling, namely, NIK and p100. We also observed that 919278 decreased the expression of other known CDK12-regulated genes, including CDK12 and RAD51 (Fig. 5A and fig. S6A). Moreover, the dual CDK7 and CDK12 inhibitor THZ1 phenocopied 919278. Similarly, targeting CDK12 with specific siRNAs also reduced the abundances of MAP3K14 and NFKB2 mRNAs as measured in separate qPCR and RNA-seq experiments.

We also found that 919278 and THZ1 reduced the extent of phosphorylation of Ser2 in the CTD of RNA Pol II, a modification that is important for transcription of long transcripts. However, the extent of phosphorylation of Ser2 in the CTD of RNA Pol II was not increased in response to TWEAK, neither were the abundances of other CDK12-dependent transcripts (CDK12 and RAD51) induced in response to TWEAK (Figs. 3A and 5A). In addition, TNF-α did not induce MAP3K14 transcription (Fig. 5D), suggesting that the mechanism for CDK12-dependent MAP3K14 transcription is specific to the noncanonical NF-κB pathway. Although stimuli of noncanonical NF-κB signaling induced MAP3K14 and NFKB2 expression, this was largely independent of NIK activity, as evidenced by the small decreases in MAP3K14 and NFKB2 mRNA abundance that occurred in response to the NIK inhibitor Amgen16 (Fig. 5, C and D). Thus, we postulate that the increase in the abundances of MAP3K14 and NFKB2 mRNAs in response to TWEAK may be due to changes in the subcellular localization of proteins required for transcription (for example, CDK12) or the composition of transcriptional complexes.

In addition, we confirmed that the noncanonical pathway components upstream of NIK were intact and functional in cells treated with 919278. For example, the cell surface abundance of FN14 was not altered in response to 919278, and the total abundance of cIAP1 decreased after treatment with TWEAK, indicating that receptor-proximal noncanonical NF-κB signaling remained intact. Despite the initiation of noncanonical NF-κB signaling, NIK protein did not accumulate in TWEAK-stimulated U-2 OS cells in the presence of 919278 (Fig. 6, A and B). As a result, cells without detectable NIK protein had a reduced ability to process p100 to p52. These data suggest that de novo transcription of MAP3K14 regulated by the CDK12-mediated phosphorylation of RNA Pol II is required for appropriate ligand-induced noncanonical NF-κB signaling.

On the basis of these collective findings, we propose the following model for noncanonical NF-κB regulation by CDK12 (Fig. 7). In the basal state, CDK12 acts (possibly in concert with CCNK) to phosphorylate key serine residues in the CTD of RNA Pol II, thereby enhancing the ability of the RNA Pol II complex to extend the length of mRNA transcripts. As a result, long transcripts, such as MAP3K14 and NFKB2 mRNAs, are generated and translated into proteins. In the absence of stimulation, NIK proteins are rapidly degraded by the proteasome, preventing activation of the noncanonical NF-κB pathway. However, in the presence of noncanonical NF-κB pathway stimuli, NIK protein accumulates, triggering the processing of p100 to p52. Thus, Rel-p52 can translocate to the nucleus and bind to κB elements, thereby regulating NF-κB–responsive gene transcription. Note that stimuli that trigger noncanonical NF-κB signaling initiate the canonical NF-κB pathway as well (22) and that canonical pathway stimulation alone can increase the transcription of NFKB2 (23). These NF-κB transcription factors recruit transcriptional machinery to κB sites, and our results demonstrate that stimulation of canonical and noncanonical NF-κB signaling led to an increase in NFKB2 mRNA abundance, which is consistent with previous reports. In contrast, MAP3K14 mRNA transcription was induced only in response to stimuli of noncanonical NF-κB signaling and was not affected by direct NIK inhibition. This suggests that the factors responsible for recruiting transcriptional machinery to the MAP3K14 locus differ from those involved at the NFKB2 locus. Regardless, initiation of transcription of both MAP3K14 and NFKB2 converged on CDK12, exemplifying the importance of CDK12 in noncanonical NF-κB pathway regulation.

Fig. 7 Model of noncanonical NF-κB signaling pathway and the effect of 919278.

Under basal (unstimulated) conditions, the CDK12/CCNK complex phosphorylates RNA Pol II, leading to the generation of MAP3K14 and NFKB2 mRNA and their translation into NIK and p100 protein, respectively. NIK associates with a TRAF2/TRAF3/cIAP complex, resulting in NIK ubiquitylation and degradation by the proteasome. In the presence of activating ligands (stimulated), CDK12/CCNK-dependent transcription of MAP3K14 and NFKB2 increases, and NIK dissociates from the TRAF2/TRAF3/cIAP complex, resulting in NIK accumulation and activation. NIK phosphorylates IKKα, which in turn phosphorylates p100. p100 is modified with ubiquitin moieties, leading to partial degradation of its C terminus and loss of its nuclear export signal. Then, p52 and its other NF-κB (Rel homology) binding partner translocate to the nucleus. In the presence of 919278 (stimulated + 919278), ligand binding to the receptor triggers proteasomal degradation of the cIAP1/TRAF complex. However, CDK12/CCNK-mediated RNA Pol II Ser2 phosphorylation is reduced, resulting in “paused” RNA Pol II and subsequently fewer NFKB2 and MAP3K14 transcripts. Without de novo synthesis of MAP3K14 mRNA, NIK cannot accumulate. Therefore, p100 is unprocessed, and this inactive precursor of p52 remains in the cytoplasm.

Similar to reports of CDK12 knockdown (13) and pharmacological inhibition (15), we found that the presence of 919278 results in the reduced phosphorylation of Ser2 in the RNA Pol II CTD, thus limiting full read-through and transcription of MAP3K14 and NFKB2. Because CDK12 is required for even basal amounts of MAP3K14 and NFKB2 mRNAs, inhibition of CDK12 prevented NIK protein accumulation and blocked noncanonical NF-κB activation. Further study will be required to fully understand the interactions between CDK12, the RNA Pol II complex, and the transcription factors that act to regulate the transcription of CDK12-dependent genes. Together, our observations add two genes to the list of genes known to be regulated by CDK12 and suggest a new therapeutic target to inhibit the noncanonical NF-κB signaling pathway.

MATERIALS AND METHODS

Cell culture and stimulations

For optimal cell performance, U-2 OS cells (American Type Culture Collection HTB-96) were maintained on a strict 2-2-3 passaging schedule in tissue culture–treated flasks at 7 × 106 cells per T150 flask and 4 × 106 cells per T150 flask in Dulbecco’s modified Eagle’s medium (DMEM) with 10% heat-inactivated fetal bovine serum (FBS) and 1× penicillin/streptomycin. Cells were cultured at 37°C with 5% CO2.

Reagents and antibodies

Antibodies against the following proteins were used for immunofluorescence: p52/p100 (Millipore, 05-361), p65 [Cell Signaling Technology (CST), 8242], Alexa Fluor 488–conjugated goat anti-mouse immunoglobulin G (IgG; CST, 4408), and Alexa Fluor 594–conjugated goat anti-rabbit IgG (CST, 8889). Antibodies and dilutions for Western blotting analysis are as follows: 1:1000 for p-Pol II CTD (Ser2) (Abcam, ab5095), p-Pol II CTD (Ser5) (CST, 13523), p-Pol II CTD (Thr4) (CST, 14934), and p-Pol II CTD (clone 4H8) (CST, 2629); 1:250 for NIK (CST, 4994); 1:1000 for cIAP1 (CST, 7065) and p100/p52 (Millipore, 05-361); 1:10,000 for β-actin (CST, 3700); and 1:10,000 to 1:20,000 for the secondary antibodies anti-rabbit–horseradish peroxidase (HRP; CST, 7074), anti-mouse-HRP (CST, 7076), and donkey anti-rabbit or donkey anti-mouse IRDye 800CW or IRDye 680RD from LI-COR. DMSO was purchased from Santa Cruz Biotechnology (sc-358801). Compounds for validation were resynthesized at Biogen. Actinomycin D was purchased from Sigma (A4262) and used at 1.59 μM. Anti-human LTβR bispecific antibody (BS-1), recombinant human TWEAK-human Fc (TWEAK), recombinant human CD40L, and the mouse anti-human FN14 antibody P4A8 were generated in-house at Biogen. Recombinant TNF-α was purchased from CST (8902). LIVE/DEAD Fixable Aqua Dead Cell Stain was used and purchased from Life Technologies (L34966).

p52 nuclear translocation compound screening assay

To identify compounds that inhibit the noncanonical NF-κB pathway, we screened 156,000 compounds at BioFocus/Charles River Laboratories using a p52 translocation imaging assay in U-2 OS cells. U-2 OS cells were trypsinized, washed, and plated at 1250 cells per well in a final volume of 20 μl of DMEM (Thermo Fisher Scientific) supplemented with GlutaMAX (Thermo Fisher Scientific) and 10% FBS. Plates were placed in an incubator at 37°C and 5% CO2 overnight. Cells were washed twice with serum-free DMEM using a BioTek Select 405 plate washer. A volume of 20 μl of serum-free medium was added to each well. U-2 OS cells were pretreated with the appropriate compounds (at 10 μM) for 30 min before being stimulated. Cells were stimulated with an anti-LTβR agonist antibody (Biogen) or TWEAK (Biogen) at a final concentration of 20 ng/ml for 4 hours in an incubator at 37°C and 5% CO2. The medium was removed, and the cells were fixed with 4% paraformaldehyde (PFA; Affymetrix #19943) at room temperature for 15 min. The cells were washed three times using a plate washer with phosphate-buffered saline (PBS) containing 0.05% Tween 20 (PBST) and then were blocked and permeabilized overnight in 20 μl of blocking buffer (5% goat serum, 0.3% Triton-X 100, and PBS). Plates were washed, and then, a primary antibody (anti-p52 at 1:1000; Millipore, 05-361) was added to each well and incubated for 1 hour at room temperature. Plates were washed and then incubated with secondary antibody (Alexa Fluor 488–conjugated goat anti-mouse IgG; 1:1000) and nuclear stain (Hoechst 33342; 1:5000) for 1 hour at room temperature. Plates were scanned, and images were collected with an OPERA HTS imaging system (PerkinElmer) and a 10× air objective. Images were then analyzed with Harmony software (PerkinElmer) by quantifying the amounts of nuclear and cytoplasmic transcription factor. Lead molecules from the primary screen were counterscreened in a TNF-α–induced p65 translocation assay.

p65 nuclear translocation assay

For the TNF-α–induced p65 translocation assay, U-2 OS cells were stimulated for 30 min with TNF-α (10 ng/ml; CST). The cells were stained with anti-p65 antibody (1:400; CST) and then with Alexa Fluor 594–conjugated goat anti-rabbit IgG (1:1000).

Compound coupling

For chemoproteomics competition experiments, a total of nine drug concentrations in the range of 0 to 30 μM in triplicate were used. Affinity beads were generated by mixing four different probe-coupled beads (pb-042, pb-137, pb-645, and pb-VI) in equal volumes. The structures and names of the probes used are shown in fig. S2. These probes were selected on the basis of their broad selectivity for kinases. Probes were immobilized on Sepharose beads through covalent linkage through the primary amine. One milliliter of N-hydroxysuccinimide (NHS)–activated Sepharose and the compound of interest (2 μmol/ml) were equilibrated in DMSO. Triethylamine (15 μl) was added to start the coupling reaction, and the mixture was incubated on an end-over-end shaker for 16 to 20 hours in the dark. Free NHS groups on the beads were blocked by adding 50 μl of aminoethanol, and the mixture was incubated on an end-over-end shaker for 16 to 20 hours in the dark. Coupled beads were washed and stored in 2-propanol at 4°C in the dark. The coupling reaction was monitored by high-performance LC.

Affinity pulldowns

The compound 919278 at planned concentrations was prepared in DMSO, and 5 μl was mixed with 50 ml of serum-free cell culture medium and added to a 15-cm culture plate containing U-2 OS cells at >95% confluence. Drug treatment was allowed for 45 min in a cell culture incubator. For TWEAK-stimulated cells, cell culture medium was replaced with fresh medium containing TWEAK (20 ng/ml) and the compound of interest, and incubation was allowed for an additional 4 hours in the cell culture incubator. Cells were washed twice with ice-cold PBS before being lysed on the plate with 300 μl of lysis buffer [50 mM tris-HCl (pH 7.5), 5% glycerol, 1.5 mM MgCl2, 150 mM NaCl, 20 mM NaF, 1 mM Na3VO4, 1 mM dithiothreitol (DTT), 5 M calyculin A, 0.8 % NP-40, and a protease inhibitor cocktail]. Cell lysates were clarified by centrifugation, and the protein concentration was measured using a Bradford assay. Cell lysate volume was adjusted to give a final concentration of 0.4% NP-40. The probe-coupled beads suspension (100 μl, 50% slurry) was added and incubated for 30 min at 4°C with rotations. A second pulldown step was performed for the control samples using fresh probe-coupled beads. This was performed to calculate the protein depletion factor. Upon completion of the incubation, the affinity beads were pelleted through centrifugation and sequentially washed with wash buffer #1 [50 mM tris-HCl (pH 7.5), 5% glycerol, 1.5 mM MgCl2, 150 mM NaCl, 20 mM NaF, 1 mM Na3VO4, 1 mM DTT, and 0.4% NP-40] and wash buffer #2 [50 mM tris-HCl (pH 7.5), 5% glycerol, 1.5 mM MgCl2, 150 mM NaCl, 20 mM NaF, 1 mM Na3VO4, 1 mM DTT, and 0.2% NP-40]. Proteins bound to the affinity beads were eluted with a sample buffer containing 2% lithium dodecyl sulfate (LDS) and reduced at 50°C for 30 min. Samples were then resolved on a 4 to 12% NuPAGE gel for about 0.5 cm to remove detergent and other buffer agents before being subjected to in-gel tryptic digestion.

In-gel digestion and LC-MS/MS analysis

In-gel digestion was performed manually or on the DigestPro in a 96-well plate format. Gel bands were first cut and diced into ~1-mm cubes and then destained with 50:50 acetonitrile/50 mM NH4HCO3 solution, reduced with DTT, and alkylated with iodoacetamide, which was followed by tryptic digestion overnight at 37°C. The tryptic peptides were extracted into 40:60 acetonitrile/0.1% formic acid solution and dried in a SpeedVac. The dry peptide extract was reconstituted in 22 μl of 2% acetonitrile/0.2% formic acid solution and analyzed on a one-dimensional nanoLC-MS/MS platform using a standardized 110-min method. Peptides were separated on a C18-AQ column (75 μm × 20 cm; Reprosil-Pur C18-AQ, 1.9 μm) at 300 nl/min, analyzed on a QExactive mass spectrometer in data-dependent acquisition mode with MS1 at a 35,000 resolution and MS2 at a 17,500 resolution, respectively.

Data, database search, process, and IC50 and Kd value calculations

MS data were first subjected to a quality-control check with in-house developed software and were subsequently searched against the Swiss-Prot human database using Andromeda integrated in Maxquant with a mass tolerance of 20 ppm (MS1) and 4.5 ppm (MS2). The protein identification and concentration were directly reported out from MaxQuant and then further processed by an in-house chemoproteomics pipeline for IC50 calculation. The competitive chemoproteomics experiment result is presented as dose-response curves (normalized protein amount versus drug concentration) and the IC50 value representing the concentration of compound that was required for 50% inhibition of detected proteins. The normalized protein amount is a ratio of the protein concentration at a given 919278 concentration to that in the DMSO control. An in-house developed R program was used to select the best curve-fitting model from four models [LL.3 (three-parameter logistic), LL.4 (four-parameter logistic), W1.3 (three-parameter Weibull), and W1.4 (four-parameter Weibull)] to derive an IC50 value for each detected protein. When a depleting factor, computed as intensity in the second pulldown/intensity at the first pull down from control samples, became available, Kd was calculated from the IC50 value by multiplying it by the depleting factor (Kd = IC50 × depleting factor). A combination of two criteria, (i) a curve-fitting P value < 1 × 10−6 and (ii) an IC50 value < 1 μM, was used to select target(s) for 919278.

siRNA-mediated knockdowns

U-2 OS cells were transiently reverse-transfected with siRNA (final concentration, 10 nM) and RNAiMax transfection reagent diluted in Opti-MEM (Thermo Fisher Scientific) in 384-well CellCarrier plates (PerkinElmer). MAP3K14-specific siRNA (Thermo Fisher Scientific, s17186) was used as positive control, and negative control #1 (Thermo Fisher Scientific, #AM4635) was used as negative control for the assay. The U-2 OS cells were trypsinized, washed, and plated at 1250 cells per well in a final volume of 20 μl of DMEM (Thermo Fisher Scientific) supplemented with GlutaMAX (Thermo Fisher Scientific) and 10% FBS. Plates were placed in an incubator at 37°C and 5% CO2 for 72 hours. The cells were then washed twice with serum-free DMEM using a BioTek Select 405 plate washer. A volume of 20 μl of serum-free medium was added to each well. The cells were then stimulated, fixed, and stained as described earlier for the p52 and p65 nuclear translocation assays.

Transcriptome data analysis

Reads were aligned to the reference genome (hg19) using STAR aligner (24). Quality control for the sequence alignment involved the analysis of sequence quality, GC content, and 5′-to-3′ gene body coverage (table S7). An outlier detection absolute Z score of >2 was applied on overall sequencing quality score, 5′ coverage, 3′ coverage, mean_GC content, duplication rate, and mean_ and mapped percentage. Samples with absolute Z scores of >2 would have been discarded, which did not apply to this study. Aligned reads were counted against gene model annotation (Gencode version 18) to obtain expression values by using featureCounts (25). DESeq2 (26) was used for gene expression normalization. Regularized log transformation function transformed the count data to the log2 scale in a way that minimized differences between samples for rows with small counts and that normalized with respect to library size. These were the values used to obtain clustering and principal components analysis results for biological quality control and downstream differential analysis. The DESeq2 generalized linear model was used for differential analysis (comparisons of treatments versus DMSO with or without TWEAK stimulation at 4 and 24 hours). A DEG signature was defined by using the following criteria: a false discovery rate of <0.05 and an absolute fold change of >2. Pathway enrichment was performed by applying the Hyper Geometric test on DEGs against canonical signaling pathways defined in MetaCore (Thomson Reuters).

Reverse transcription PCR assays

For concentration-response and time-course experiments, 10,000 U-2 OS cells per well were seeded in a 96-well format the day before treatments, which were performed in triplicate. Cell lysis with concurrent deoxyribonuclease digestion followed by complementary DNA (cDNA) synthesis was performed with a Cells-to-CT kit (Thermo Fisher Scientific) according to the manufacturer’s protocol. Reverse transcription PCR (RT-PCR) analysis of 10-fold diluted cDNA in 10-μl reaction volumes was performed with TaqMan Fast Advanced Master Mix (Thermo Fisher Scientific) on a QuantStudio7 RT-PCR system (Thermo Fisher Scientific) with the following cycling parameters: 50°C for 2 min, 95° for 20 s, and then 40 cycles of 95°C for 1 s and 60°C for 20 s. Quadruplicate RT-PCR amplification was done for each sample. Target mRNA abundance relative to that of the mRNA of a reference gene (Actb or GAPDH) was calculated by the ΔΔCt method. For B cell experiments, 400,000 to 800,000 peripheral human B cells (STEMCELL 70023, lots: 1604070067, 1602290181, and 1604150127) were seeded per well in a 96-well round-bottom format on the day of stimulation. Cells were pretreated for 30 min with DMSO or the compound of interest before being stimulated with CD40L (500 ng/ml) for 4 hours. RNA extraction was performed with a Qiagen RNA Micro Plus kit, and cDNA synthesis was performed with a High-Capacity cDNA Reverse Transcription kit (Applied Biosystems) according to the manufacturer’s protocol. RT-PCR analysis of fivefold diluted cDNA in 10-μl reaction volumes was performed with TaqMan Gene Expression Master Mix (Applied Biosystems) on a QuantStudio7 RT-PCR system (Thermo Fisher Scientific) with the following cycling parameters: 50°C for 2 min, 95° for 10 min, and 50 cycles of 95°C for 15 s and 60°C for 60 s. Triplicate RT-PCR amplification was done for each sample. Target mRNA abundance relative to that of the mRNA of a reference gene (Actb or GAPDH) was calculated by the ΔΔCt method. The following TaqMan probes (Thermo Fisher Scientific) were used: CDK12, Hs00212914_m1; MAP3K14 (NIK), Hs01089753_m1; NFkB2, Hs01028901_g1; RAD51, Hs00947967_m1; and ACTB (β-actin), 4352935.

Preparation of U-2 OS cell lysates for protein detection

For Western blotting analysis, cells were plated at 0.5 × 106 cells per well in a six-well plate and incubated for 1 day at 37°C, 5% CO2. Before stimulation, the cells were washed with serum-free DMEM, in which they were maintained for the duration of the stimulations. The compounds of interest or DMSO were added 30 min before the cells were stimulated with TWEAK (20 ng/m). Four hours later, the cells were washed with PBS and then lysed in LDS buffer (Invitrogen, NP0007), reducing agent (Invitrogen, NP0009), and 1× protease and phosphatase inhibitors (CST, 5871 and 5870). Samples were collected, processed with QiaShredder tubes at 2000g for 2 min, and then heated at 70°C for 10 min or 95°C for 5 min.

Western blotting analysis

Each sample (10 μl) was loaded onto a 4 to 12% bis-tris, 17-well protein gel (Invitrogen, NP0329BOX) and resolved with 1× MOPS Buffer (Invitrogen, NP0001) under reducing conditions with antioxidant (Invitrogen, NP0005) at 200 V for 50 to 60 min. Proteins were then transferred to a 0.2- or 0.45-μm nitrocellulose membrane (Invitrogen, LC2000) in a wet transfer with 1× transfer buffer (Invitrogen, NP0006), 10% methanol (Fisher, A452), and antioxidant for 1 hour at 400 mA or 30 V for 2 hours. Blots were then blocked while shaking in 5% milk (American Bioanalytical, AB10109-01000) in PBST or Li-COR Blocking Buffer (927-50000) for at least 1 hour at room temperature before being incubated overnight at 4°C with primary antibody while shaking. After washing, the blots were incubated for 1 hour with secondary antibody at room temperature while shaking. All washes were performed in 1× PBST. After incubation with secondary antibody, the blots were washed three times with PBST and once with PBS. Blots were developed with an enhanced chemiluminescence (ECL) substrate (Pierce, 32106) or West Dura ECL Substrate (Pierce, 34076) using Biomax light film (Carestream Kodak, Z370371) and the Amersham Imager 600 or the Odyssey CLx Imaging System for fluorescently conjugated antibodies.

Flow cytometric analysis of cell surface FN14 abundance

For evaluation of FN14 surface abundance, U-2 OS cells were plated at 0.1 × 106 cells per well in a 12-well plate and incubated overnight at 37°C and 5% CO2. Before stimulation, the cells were washed with serum-free DMEM and were maintained in serum-free DMEM for the duration of the experiment. Compounds (10 μM for Amgen16, 919278, and 702697 or 1 μM for THZ1) or DMSO was added 30 min before the cells were treated with TWEAK (20 ng/ml) or medium alone. The cells were then incubated an additional 30 min. The cells were washed with PBS and harvested with TrypLE Express from Gibco (12604-021) Cells were collected and pelleted at 425g for 2 min before being stained with P4A8 (1 μg/ml; mouse anti-human FN14 antibody) in fluorescence-activated cell sorting (FACS) buffer with LIVE/DEAD Fixable Aqua Dead Cell Stain (1:600) for 20 min on ice. The cells were washed twice with FACS buffer and pelleted as described earlier. Cells were incubated with secondary anti-mouse IgG antibody (1:1000) for 20 min on ice. Cells were then washed twice and resuspended in 2% PFA overnight at 4°C in the dark. The following day, the cells were washed in FACS buffer and analyzed with an LSR II flow cytometer (five-laser). Cell gating is shown in fig. S7 with representative histograms shown in fig. S7.

SUPPLEMENTARY MATERIALS

www.sciencesignaling.org/cgi/content/full/11/541/eaam8216/DC1

Text S1. Synthesis of 919278.

Text S2. Synthesis of 702697.

Fig. S1. The IKKβ inhibitor ACHP inhibits TNF-α–induced p65 nuclear translocation in U-2 OS cells.

Fig. S2. Overview of the chemoproteomics workflow.

Fig. S3. Docking of 919278 in CDK12.

Fig. S4. Compound 919278 reduces the phosphorylation of Ser2, but not Ser5, in the RNA Pol II CTD.

Fig. S5. Hierarchical clustering of Spearman correlation coefficients demonstrates that compound 919278 has similar effects to CDK12-specific siRNA.

Fig. S6. Compound 919278 inhibits the noncanonical NF-κB–induced expression of MAP3K14 and NFKB2.

Fig. S7. Compound 919278 does not alter FN14 abundance or cell viability but reduces p100 processing.

Table S1. Reaction Biology kinase panel (enzymatic) with a racemic mixture of 919278 and 702697.

Table S2. DiscoverX kinome panel.

Table S3. ActivX profiling of U-2 OS cells treated with compound 919278 or compound 702697.

Table S4. List of all of the endogenous kinases detected and quantified in the U-2 OS cell proteome during the chemoproteomics studies.

Table S5. Effect of CCNK knockdown on p52 nuclear translocation.

Table S6. List of DEGs in response to TWEAK.

Table S7. Quality control statistics for the RNA-seq analysis.

REFERENCES AND NOTES

Acknowledgments: We would like to acknowledge the University School of Medicine’s Department of Pharmacology and Experimental Therapeutics for providing support (to J.E.A. and K.L.H.) as PhD students in their program. Funding: All funding was provided by Biogen. Author contributions: K.L.H. prepared the manuscript and designed and conducted the experiments. E.C.H., E.C.-M., and M.L.O. prepared the manuscript and designed experiments. D.K., B.B., J.E.A., M.B., A.G.C., E.V., K.A.E., and W.Z. designed and conducted experiments. J.F., B.G., and K.L. designed experiments and analyzed data. B.L., G.B., T.C., A.B.-C., B.H., T.J., T.M.-D., P.M., R.W., N.A., A.B., C.L., and P.J. designed experiments and provided scientific guidance. Competing interests: The authors are or were employees of Biogen and hold stock. Data and materials availability: RNA-seq data were deposited at the National Center for Biotechnology Information as Gene Expression Omnibus number: GSE113926. All other data needed to evaluate the conclusions in the paper are present in the paper or the Supplementary Materials.
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