Compounds targeting the circadian clock have been identified as potential treatments for clock-related diseases, including cancer. Our cell-based phenotypic screen revealed uncharacterized clock-modulating compounds. Through affinity-based target deconvolution, we identified GO289, which strongly lengthened circadian period, as a potent and selective inhibitor of CK2. Phosphoproteomics identified multiple phosphorylation sites inhibited by GO289 on clock proteins, including PER2 S693. Furthermore, GO289 exhibited cell type–dependent inhibition of cancer cell growth that correlated with cellular clock function. The x-ray crystal structure of the CK2α-GO289 complex revealed critical interactions between GO289 and CK2-specific residues and no direct interaction of GO289 with the hinge region that is highly conserved among kinases. The discovery of GO289 provides a direct link between the circadian clock and cancer regulation and reveals unique design principles underlying kinase selectivity.

We aimed to expand the chemical tools enabling dissection and control of the circadian clock system for a better understanding of the molecular mechanism. Here, we report the discovery of a new period-lengthening compound, GO289, whose target protein was identified as CK2 through an affinity-based proteomics approach. CK2 is one of the most well-studied protein kinases because of its ubiquitous expression, pleiotropy, and constitutive activity. On the basis of its major function in cell growth and apoptosis, CK2 is known as one of the key players in the pathogenesis of cancer ( 27 , 28 ). Small-molecule inhibitors of CK2, such as TBB, DMAT, and CX-4945, have played important roles in understanding the functions of this kinase, but their selectivity is not optimal as either tool molecules or potential therapeutic agents ( 29 – 31 ). In mammals, the role of CK2 in circadian period regulation is less well characterized compared to CKI, partly because of the lack of genetic models due to lethality and the lack of selective chemical tools. The discovery of GO289 overcomes some of these issues. Through its high potency and selectivity, GO289 enabled manipulation of clock protein phosphorylation and cancer cell growth. Furthermore, x-ray crystallographic studies revealed the molecular basis underlying the potency and selectivity of GO289 through a binding modality relatively distinct from most known protein kinase inhibitors.

Perturbations of clock function by genetic mutations or environmental factors, such as shift work, have been implicated in sleep disorders, cancer, and cardiovascular and metabolic diseases in humans and animal models ( 1 – 3 ). Therefore, identification of small molecules modulating circadian clock function will provide new opportunities for treatments of clock-related disorders. Cell-based high-throughput chemical screening approaches have led to the discovery of a number of compounds that affect circadian rhythms ( 11 , 12 ). Consistent with the functional importance of CKIδ/ε, multiple compounds that inhibit CKI activity have been identified to lengthen circadian period ( 13 – 17 ). Among these, well-known inhibitors for a number of different kinases [cyclin-dependent kinase, Cdc2-like kinase (CLK), p38 mitogen-activated protein kinase, c-Jun N-terminal kinase, casein kinase 2 (CK2), and vascular endothelial growth factor receptor] have been shown to be potent inhibitors of CKI ( 14 ), highlighting a key but complex issue of kinase inhibitor selectivity ( 18 ). The development of new CKI inhibitors with higher specificity through target-based approaches ( 19 , 20 ) or unbiased approaches ( 15 , 16 ) enabled the conditional manipulation of CKI and modulation of circadian period in CKIε tau mutant mice ( 21 ). We further discovered a unique chemical modulator of CRY by screening for period-changing compounds followed by affinity-based target deconvolution. This compound, KL001, inhibits proteasomal degradation of CRY, lengthens circadian period in a variety of cells and tissues, and blocks glucagon-dependent induction of gluconeogenesis in cultured hepatocytes ( 22 ). Consistent with these observations, a bioavailable derivative of KL001 exhibits antihyperglycemic activity in db/db diabetic mice ( 23 ). Moreover, small-molecule agonists of REV-ERB and ROR improve energy homeostasis ( 24 , 25 ), and REV-ERB agonists impair cancer growth to improve survival in a mouse model of glioblastoma ( 26 ). The discovery of new compounds that target the circadian system offers a previously unidentified strategy to control clock-related processes, and there are still numerous clock-modulating compounds whose target proteins have not yet been identified.

In addition to transcriptional regulation, posttranslational modification of clock proteins forms an indispensable layer necessary for circadian rhythmicity. Most clock proteins undergo rhythmic phosphorylation ( 4 ), and the functional importance of clock protein phosphorylation has been proven by spontaneous gene mutations, resulting in alterations of circadian period. The tau mutant hamster with short-period behavioral rhythms has a missense mutation in the casein kinase I (CKI) ε gene ( 5 ), and human familial advanced sleep phase (FASP) with early sleep times has been attributed to missense mutations in the PER2 and CKIδ genes ( 6 , 7 ). CKIε and CKIδ phosphorylate PER to trigger proteasomal degradation, and the tau and FASP mutations lead to faster degradation of PER, consistent with the short-period phenotype ( 8 , 9 ). Phosphorylation of CRY1 also regulates circadian period through degradation-dependent and degradation-independent pathways ( 10 ). To fully understand and characterize the phosphorylation network controlling the circadian clock, it is necessary to identify the phosphorylation sites and determine the responsible kinases for each clock protein.

The circadian clock is an intrinsic timekeeping mechanism that controls daily rhythms of many physiological processes, including sleep/wake behavior, body temperature, hormone secretion, energy metabolism, and the cell cycle. Circadian rhythms are generated in a cell-autonomous manner, and within each cell, clock genes form transcriptional regulatory networks. The transcription factors CLOCK and BMAL1 activate expression of Period (Per1 and Per2) and Cryptochrome (Cry1 and Cry2) genes. After translation, complex formation, and nuclear localization, PER and CRY proteins inhibit CLOCK-BMAL1 function, resulting in rhythmic gene expression. In parallel, the Bmal1 gene is regulated by nuclear hormone receptors REV-ERB and ROR, whose gene expression is controlled by the CLOCK-BMAL1 complex to form an interconnected feedback loop ( 1 – 3 ).

RESULTS

Identification of a new compound that lengthens circadian period To identify small molecules that modulate the cell-autonomous function of the circadian clock, we conducted a cell-based chemical screen using human U2OS osteosarcoma cells harboring a Bmal1 promoter-luciferase (Bmal1-dLuc) reporter. Confluent cells were treated with an in-house collection of drug-like compounds, and circadian luminescence rhythms were measured. We found a number of compounds with different chemical scaffolds that strongly changed the period of circadian rhythms (15, 16, 22). In this study, we focused on the guaiacol derivative, GO289 (Fig. 1A), with no known biological activity. This compound caused dose-dependent lengthening of circadian period not only in Bmal1-dLuc reporter cells but also in Per2-dLuc reporter cells with a phase opposite to that of Bmal1-dLuc (Fig. 1B). GO289 also lengthened periods in cells differentiated from embryonic stem (ES) cells of Per2::LucSV knock-in mice harboring a PER2-LUC fusion protein reporter (Fig. 1C) and in lung explants from Per2::Luc mice (fig. S1A). These results indicate that GO289 reproducibly causes strong period lengthening regardless of the reporter or cell type in human and mouse. Fig. 1 GO289 lengthens circadian period. (A) Chemical structure of GO289. (B and C) Effect of GO289 on circadian rhythms in Bmal1-dLuc and Per2-dLuc U2OS cells (B) and cells differentiated from Per2::LucSV knock-in ES cells (C). Luminescence rhythms were monitored in the presence of various concentrations of GO289 and shown in the left (Bmal1-dLuc) and middle (Per2-dLuc) panels of (B) and the left panel of (C) (mean of n = 4). Period changes compared to a dimethyl sulfoxide (DMSO) control are plotted in the right panel of (B) and (C) (n = 4). ****P < 0.0001 and ***P < 0.001 against the DMSO control. (D) General synthetic scheme for GO289 derivatives. (E) Period-lengthening activity of GO289 derivatives. Luminescence rhythms of Bmal1-dLuc cells were monitored in the presence of various concentrations (threefold, 12-point dilution series) of GO289 derivatives (n ≥ 2), and the concentration required for half-maximal period lengthening is shown as logEC 50 . Modified part of the compound is shown in color. C4 and C3 positions of the benzene ring at R6 correspond to the para and meta positions, respectively. (F) Summary of the SAR study. We previously demonstrated that the period-lengthening compounds longdaysin and KL001 inhibited CKI activity and CRY degradation, respectively (15, 22). In contrast, GO289 showed minor effects on CKIδ and CKIα activity in vitro (fig. S1B) and no stabilization of CRY1 in a cell-based degradation assay (fig. S1C), suggesting an alternative mechanism of action from previously known clock modulators.

Structure-activity relationship of GO289 GO289 consists of a triazole structure linked to bromoguaiacol, methyl thioether, and phenyl groups (Fig. 1A). To investigate functional sites of GO289 responsible for its effect on circadian period, we performed a structure-activity relationship (SAR) study. The general synthetic scheme for GO289 derivatives is shown in Fig. 1D. Arylhydrazides (I) were converted to potassium aroyl dithiocarbazates and then to amino triazole (II) by a ring-closure reaction with hydrazine and subsequent alkylation (32). Imine formation of II with aryl aldehyde furnished the core structure of GO289 (III). Each group was modified by substituting I, alkyl reagent, and aryl aldehyde. We analyzed the effect of GO289 derivatives on circadian period in Bmal1-dLuc U2OS cells (Fig. 1E). Both triazole and bromoguaiacol groups were required for the activity, as either group alone did not show any effect on period (1, 2). Removal of all three substituents in the bromoguaiacol (Br, hydroxy, and methoxy) caused a complete loss of activity (3). Addition of bulkier substituents also resulted in a severe reduction in period-lengthening activity (4, 5, 7, 8, 9, 10), with the exception of acetylation of the hydroxy group that slightly increased activity (6). Addition of groups at an unsubstituted meta or ortho position also led to decreased activity (11, 12, 13), indicating that the bromoguaiacol cannot be modified. In contrast, removal of either the methyl thioether group or the phenyl group was tolerated (14, 15), although removal of both groups caused a severe reduction in activity (16). Addition of bulkier groups to the methyl thioether group strongly reduced activity (17, 18, 19). Similarly, modification of the phenyl group at the meta position decreased activity, while addition at the para position had little effect (20, 21, 22, 23, 24, 25). Together, the SAR analysis indicated that the bromoguaiacol is essential for activity, and the para position of the phenyl group is amenable for modification (Fig. 1F).

Target identification of GO289 To identify molecular targets of GO289, we used an affinity-based proteomics approach. On the basis of the SAR data (Fig. 1E), we attached a tetraethylene glycol linker to the para position of the phenyl group on GO289 (Fig. 2A). The compound GO457 retained significant period-lengthening activity (Fig. 2B) and therefore was used as an affinity probe. Proteins interacting with an agarose conjugate of GO457 were affinity-purified and analyzed by liquid chromatography–tandem mass spectrometry (LC-MS/MS). From two independent experiments, CK2α (CSNK2A1), CK2α′ (CSNK2A2), CK2β (CSNK2B), and GSK-3β (GSK3B) were highly enriched with the affinity probe. These interactions were blocked by free GO289 (Fig. 2C), indicating that they are candidates for GO289-target proteins. All of these proteins are known to be involved in regulating circadian period, as knockdown of CK2α, CK2α′, and CK2β lengthens period (33, 34) and GSK-3β knockdown causes period shortening (13) in U2OS cells. The spectral numbers for CK2α and CK2α′ under competition (−) conditions were much higher than those for CK2β and GSK-3β, suggesting that CK2α and CK2α′ are the primary targets of GO289 (Fig. 2C). CK2 is a Ser/Thr kinase, whose catalytic subunits (CK2α and CK2α′) and regulatory subunit (CK2β) exist as monomers, as well as α 2 β 2 tetramers. By immunoblotting with specific antibodies against CK2α and CK2β, we confirmed their enrichment with the affinity probe and competition by free GO289 (Fig. 2D). Furthermore, recombinant CK2 (α 2 β 2 tetramer) also interacted with the affinity probe, and this interaction was blocked by increasing concentrations of free GO289 (Fig. 2E), indicating a direct interaction of GO289 with CK2. Fig. 2 GO289 interacts with CK2. (A) Chemical structure of GO457. (B) Effect of GO457 on circadian period in Bmal1-dLuc U2OS cells. ****P < 0.0001 and ***P < 0.001 against the DMSO control (n = 4). (C) GO289-interacting proteins. Agarose-conjugated GO457 was incubated with a U2OS cell lysate in the presence of 0 and 50 μM GO289 [competition (−) and (+), respectively]. Affinity-purified proteins were analyzed by LC-MS/MS. Proteins identified by ≥3 tandem MS spectra from ≥2 unique peptides (numbers in parentheses) and showed ≥5-fold signal reduction upon competition with GO289 [ratio (+)/(−) ≤ 0.2] in the first analysis are shown (columns 2 to 4). Enrichment with the affinity probe was estimated by comparing with the MS spectra number in the input sample (columns 5 and 6). Low enrichment efficiency (recovery ≤ 10%) is indicated in italics. The result of the second analysis focusing on 20- to 55-kDa proteins is shown in columns 8 to 10. Proteins fulfilling the criteria in both analyses are indicated in red. MW, molecular weight. (D and E) Interaction of GO289 with CK2. Affinity-purified proteins from a U2OS cell lysate (D) or recombinant CK2 (E) were analyzed by immunoblotting with specific antibodies. The asterisk indicates a nonspecific protein that interacted with the probe, independent of GO289.

Potent and highly selective inhibition of CK2 by GO289 The effect of GO289 on CK2 activity was analyzed using an in vitro kinase assay. GO289 potently inhibited CK2 at a half-maximal inhibitory concentration (IC 50 ) of 7 nM (Fig. 3A). In contrast, 5 μM GO289 showed only a moderate or minor effect on the activity of 59 kinases from a variety of classes (Fig. 3B). The second most affected kinase was PIM2, with an IC 50 of 13 μM (Fig. 3C), which was >1000 times higher than that for CK2. The minor effect of GO289 on GSK-3β activity (Fig. 3B) supported the idea that GSK-3β is most likely not a direct target. Together, the protein interaction and kinase inhibition profiles indicate high potency and selectivity of GO289 toward CK2. Fig. 3 GO289 potently and selectively inhibits CK2. (A to D) Effect of GO289 on kinase activity in vitro. Activity of CK2 (A) and PIM2 (C) was analyzed in the presence of GO289 at various concentrations (n = 2). A panel of 60 kinases (B) and DYRK, HIPK, and PIM family kinases (D) were screened with 5 μM GO289 (n = 2). In (D), the effect of GO289 on multiple kinases is compared to published values for CK2 inhibitors TBB, DMAT, and CX-4945. ND, not determined. (E) Effects of CK2 inhibitors on circadian period and reporter signal intensity in Bmal1-dLuc U2OS cells. Changes in period (left) and luminescence intensity (right) compared to the DMSO control are plotted (n = 4). P values are summarized in table S3. (F and G) Effect of GO289 on cellular CK2 activity. HEK293T cells (F) or U2OS cells (G) were treated with GO289 at various concentrations for 24 hours and subjected to immunoblotting with anti-phosphorylated (anti-phospho) CK2 substrate antibody. The membrane was reprobed with anti-phospho PKA (protein kinase A) substrate and anti–β-actin antibodies (F) or stained with CBB (Coomassie Brilliant Blue) (G). Well-known CK2 inhibitors, TBB and DMAT, strongly inhibit DYRK, HIPK, and PIM family kinases, in addition to CK2 (Fig. 3D) (29). CX-4945 is also a potent CK2 inhibitor with high selectivity, yet still inhibits DYRK, HIPK, and PIM family kinases (Fig. 3D) (30, 35). In contrast, GO289 showed little effect on these other kinases, even at a concentration of 5 μM, which is 700 times higher than its IC 50 for CK2 (Fig. 3D). The IC 50 ratios of TBB, DMAT, and CX-4945 against CK2 over PIM family kinases are 5.7 (PIM3), 0.7 (PIM3), and 46 (PIM1), respectively, while that of GO289 was >1000 (PIM2) (fig. S2A). Furthermore, CX-4945 is reported to inhibit CLK family proteins (30, 31). In our assays, the IC 50 values of CX-4945 against CK2 and CLK2 were 6 and 23 nM, respectively (fig. S2, B and C), while GO289 showed no effect on CLK2 (IC 50 of >50 μM). Consistent with the lower selectivity of DMAT and CX-4945, they both caused a severe reduction in luminescence in a dose-dependent manner in our cell-based circadian assay (Fig. 3E), possibly because of cytotoxicity arising from multitarget effects. In contrast, GO289 lengthened circadian period by more than 12 hours with little effect on luminescence compared to DMAT and CX-4945. These results together highlight the potency and selectivity of GO289. We further analyzed the effect of GO289 on cellular CK2 activity by measuring phosphorylation of CK2 substrates by immunoblotting. Human embryonic kidney (HEK) 293T cells were treated with GO289, and cell lysates were analyzed using an anti-phospho CK2 substrate antibody. This antibody recognizes the CK2-consensus sequence pS/pT-D-X-E, in which pS/pT represents Ser/Thr residues phosphorylated by CK2. The antibody detected many protein substrates, and the phosphorylation signal was reduced by GO289 in a dose-dependent manner (Fig. 3F, left). In contrast, neither the CKIδ inhibitor LH846 nor the GSK-3 inhibitor CHIR-99021 altered the CK2-specific phosphorylation pattern (fig. S3). Furthermore, the signal detected by the anti-phospho PKA (protein kinase A) substrate antibody was not affected by GO289 (Fig. 3F, right), demonstrating the specificity of both GO289 and the anti-phospho CK2 substrate antibody. Inhibition of cellular CK2 activity by GO289 was also observed in U2OS cells at concentrations similar to those that affected circadian period (Fig. 3G), linking inhibition of CK2-dependent phosphorylation to period lengthening.

Regulation of clock protein phosphorylation by GO289 CK2 has been reported to regulate circadian clock oscillations across species, including Neurospora, Arabidopsis, Drosophila, and mammals. In mammals, CK2 phosphorylates PER2 and leads to nuclear accumulation (33) or degradation (36) and also phosphorylates BMAL1, which results in nuclear accumulation (37). Previous in vitro phosphorylation studies identified the CK2-dependent sites T12/S13 and S53 of PER2 and S90 of BMAL1. However, mutation of each of these sites has little effect on circadian period (33, 36, 37) compared to the prominent period-lengthening effects of GO289. We therefore conducted an extensive search for cellular CK2 phosphorylation sites in core clock proteins by inhibiting CK2 activity with GO289. HEK293T cells expressing Flag-tagged PER2, CRY1, CLOCK, and BMAL1, together with CK2α, were treated with GO289. After immunoprecipitation with an anti-Flag antibody, the clock proteins were subjected to phosphoproteomic analysis by LC-MS/MS, in which combinations of four proteases were used to obtain high-sequence coverage. In PER2, CRY1, CLOCK, and BMAL1, we identified 15, 3, 3, and 9 residues, respectively, whose phosphorylation was reduced by GO289 (Fig. 4A). Among these, S693 of PER2 exhibited the strongest signal. Because a previous study suggested that S693 or S697 of full-length PER2 was phosphorylated by both CKIδ and CK2 in vitro (33), we synthesized a peptide PER2(680-705) encompassing both of these amino acids and performed an in vitro kinase assay. This peptide was phosphorylated by CK2, but not by CKIδ (Fig. 4B). Mutation of T687, S693, and/or S697 to Ala identified S693 as the site phosphorylated by CK2 in the PER2(680-705) peptide (Fig. 4C). These observations indicate that CK2, but not CKIδ, is responsible for phosphorylation of PER2 S693 that is inhibited by GO289 in cells. In contrast, previously reported CK2 phosphorylation sites T12/S13 and S53 of PER2 were either unchanged or up-regulated by GO289 in our cell-based assay using full-length PER2 (table S1). Fig. 4 GO289 affects phosphorylation of clock proteins. (A) Phosphorylation sites inhibited by GO289. HEK293T cells expressing Flag-tagged mouse clock proteins and CK2α were treated with GO289. Immunoprecipitated clock proteins were subjected to LC-MS/MS analysis. Phosphorylation level was calculated by dividing spectra number of phosphorylated peptides with total peptides (ratio). Effect of GO289 was defined as the ratio of the GO289-treated sample to the control sample (GO289/control). Sites abundantly phosphorylated (ratio of control sample ≥ 0.1) and inhibited by GO289 (GO289/control ≤ 0.5) are shown. (B and C) Phosphorylation of PER2(680-705) peptide by CK2. (B) Activity of CK2 and CKIδ was analyzed by measuring ATP levels (n = 3 to 6). (C) Ser/Thr residues were mutated to Ala (n = 2 to 4), and ATP half-life is plotted in the right panel. ****P < 0.0001, ***P < 0.001, **P < 0.01 against no peptide control. (D) Effect of GO289 on circadian rhythms of Bmal1-Luc reporter in wild-type and Per2 knockout cells. Period changes compared to the DMSO control are plotted in the right panel (n = 3 to 4). P values are against wild-type cells. (E) Effect of GO289 on BMAL1 S90 phosphorylation. Mouse NIH-3T3 cells were treated with 10 μM GO289 for different times and subjected to immunoprecipitation/immunoblotting with specific antibodies. To evaluate the role of PER2 phosphorylation by CK2 in period regulation, we applied GO289 to Per2 knockout cells differentiated from ES cells. Period lengthening of Bmal1-Luc rhythms by GO289 was slightly enhanced in Per2 knockout cells compared to wild-type cells (Fig. 4D), supporting a functional interaction between CK2 and PER2. However, since the period-changing effects of GO289 were not abolished in Per2 knockout cells, CK2 substrates other than PER2 might also be involved in GO289-dependent changes in circadian period. Our phosphoproteomic analysis identified candidate phosphorylation sites in CRY1, CLOCK, and BMAL1, in addition to those in PER2 (Fig. 4A). As for the previously reported CK2-site BMAL1 S90, only one spectrum of the phosphorylated peptide was observed in both control and GO289-treated conditions, but immunoblot analysis using a specific antibody against S90-phosphorylated BMAL1 revealed its down-regulation by GO289 as represented by reduction of immunoreactivity (Fig. 4E). The band of S90-phosphorylated BMAL1 became broad possibly because of phosphorylation-dependent acetylation and SUMOylation of BMAL1 (38). Thus, it is likely that CK2 targets multiple protein substrates for its strong effect on circadian period, and GO289 provides an important tool to further investigate CK2-dependent phosphorylation of clock proteins and how this might regulate circadian period.

Regulation of cancer cell growth by GO289 In addition to regulating the circadian clock, CK2 plays important roles in cell growth and apoptosis, and elevated CK2 levels are associated with tumorigenesis (27, 28). Because perturbation of circadian clock function is linked to cancer (39), CK2 may be a key molecule connecting them. We therefore analyzed the effect of GO289 on cell growth in human renal cell carcinoma (RCC) cell lines, whose circadian properties have been characterized previously (40). Growth of Caki-2, A498, and 769-P cells was strongly inhibited by GO289, while that of 786-O, RCC4+vec, RCC4+VHL, A704, and ACHN cells was less affected (Fig. 5A). Similarly, CX-4945 inhibited growth of Caki-2 cells but had less of an effect in A704 and ACHN cells (fig. S4A), demonstrating cell type–dependent regulation of growth by CK2. We observed that Bmal1 reporter induction (40) correlated well with GO289 sensitivity (Fig. 5B), suggesting an interaction between clock function and CK2-dependent growth inhibition. Fig. 5 GO289 inhibits cancer cell growth. (A and C) Effect of GO289 on growth of human RCC lines (A) and mouse AML MLL-AF9 cells or normal LSK cells (C). Cell numbers are plotted in the left panel by setting the DMSO control to 1 [n = 6 in (A) and n = 3 in (C)]. For (A), pIC 50 values are plotted on the right, and P values are summarized in table S3. (B) Correlation of growth inhibition by GO289 with Bmal1 reporter induction. Degree of Bmal1 reporter induction (40) was calculated by dividing intensity of the peak with time 0 and plotted against pIC 50 values from (A). (D) Effect of GO289 on circadian period and reporter signal intensity in spleen explants of MLL-AF9 mice. Luminescence rhythms of the Per2::Luc knock-in reporter were monitored in the presence of GO289 and indicated in the top left (MLL-AF9 mice, mean of n = 11) and the bottom left (control mice, mean of n = 10 to 20). Changes in intensity (top right) and period (bottom right) compared to the DMSO control are plotted. ****P < 0.0001, **P < 0.01 against the DMSO control. We further analyzed the effect of GO289 in a mouse model of acute myeloid leukemia (AML). A screen of genes whose knockdown inhibited growth of mouse MLL-AF9 leukemia cells has been reported, and CK2α (Csnk2a1) and CK2β (Csnk2b) were identified as one of the hit genes (41). Consistent with this, we found that pharmacological inhibition of CK2 with GO289 significantly reduced growth of mouse MLL-AF9 leukemia cells, while Lin−Sca1+c-Kit+ (LSK) hematopoietic progenitor cells as a normal counterpart were much less affected (Fig. 5C). Furthermore, the human AML cell line OCI-AML3 was also sensitive to GO289 (fig. S4B). We then investigated circadian rhythms in MLL-AF9 leukemia cells that were prepared from bone marrow cells of Per2::Luc reporter mice and transplanted into nonreporter mice. We found that circadian rhythms in spleen and bone marrow explants were severely disrupted in MLL-AF9 leukemia mice compared to control mice that were transplanted with wild-type bone marrow cells from Per2::Luc reporter mice (Fig. 5D and fig. S4C). GO289 caused a severe reduction in luminescence in MLL-AF9 tissues relative to the control, consistent with its anticancer effect. Furthermore, the compound lengthened circadian period in control spleen and bone marrow explants. Together, these results demonstrated that GO289 inhibits growth of cancer cells in a cell type–dependent manner and directly links the circadian clock with cancer (see Discussion).