Chk1 Inhibition Potently Blocks STAT3 Tyrosine705 Phosphorylation, DNA-Binding Activity, and Activation of Downstream Targets in Human Multiple Myeloma Cells

This article is featured in Highlights of This Issue, p. 355

STAT3 is a transcription factor that regulates the expression of diverse genes implicated in tumorigenesis, proliferation, and survival, particularly in the case of hematologic malignancies such as multiple myeloma (1, 2). STAT3 activation has been identified in up to 50% of patients with multiple myeloma and is associated with a poor prognosis (3). STAT3 responds to diverse growth factors and cytokines (e.g., IL6) which activate receptor and non-receptor tyrosine kinases such as JAK1/2. This leads to phosphorylation of the STAT3 transactivation domain on Tyr₇₀₅, necessary for dimerization and nuclear translocation, culminating in DNA binding (4). STAT3 is also activated by G protein–coupled receptors and Toll-like receptors (4). Other pathways, for example, MEK1/2/ERK1/2 have been linked to STAT3 Ser₇₂₇ phosphorylation, which increases transcriptional activity (5, 6).

Activation of STAT3 triggers expression of multiple pro-survival and pro-proliferation genes including MCL-1, BCL-X_L, and c-Myc, among others (7). Notably, STAT3 hyperactivation has been implicated in drug resistance (8), particularly microenvironmental forms of resistance (9). While the development of inhibitors of STAT3, like other transcription factors, has been challenging, JAK inhibitors are currently approved for the treatment of patients with myelofibrosis and JAK2 mutations. Although preclinical evidence suggests a possible role for such agents in multiple myeloma (10), the existence of JAK-independent pathways of STAT3 activation may limit the effectiveness of this approach. Other strategies to disrupt STAT3 function include the use of peptidomimetics, anti-sense oligonucleotides, decoy oligonucleotides (4), or small-molecule inhibitors/degraders, for example, Stattic, atovaquone (11), SD-36 (PROTAC; ref. 12) However, drug delivery and pharmacokinetic issues continue to represent barriers to successful STAT3 targeting (13).

The Chk1 kinase represents a critical component of the DNA damage response (DDR; ref. 14). Upon genotoxic injury and development of single-strand breaks, Chk1 is activated by ATR (ataxia telangiectasia-mutated and Rad3-related kinase) and phosphorylates the Cdc25C and Cdc25A phosphatases, leading to their ubiquitination and degradation (15). This promotes inhibitory phosphorylation of cdc2 (CDK1) on Tyr₁₅ and Thr₁₄ sites, allowing cells to arrest in G₂ and repair DNA damage (16). Chk1 inhibition spares CDC25A/C, induces “inappropriate” dephosphosphorylation/activation of CDC2, loss of the intra-S-phase checkpoint, increased replication stress, and progression of cells through G₂ into mitosis, leading to mitotic catastrophe and cell death (17). In extensive preclinical studies, Chk1 inhibitors synergistically increase the antitumor activity of diverse genotoxic agents in vitro and in vivo (18), including in multiple myeloma (19), prompting the development of multiple clinical Chk1 inhibitor candidates (20). To date, essentially all Chk1 inhibitor strategies have involved combination with genotoxic drugs (21). However, results of such studies have been equivocal, perhaps reflecting the inability of this strategy to improve the therapeutic index of DNA-damaging agents (22). On the other hand, recent attention has focused on novel functions of Chk1 in neoplastic cell survival, proliferation, and DNA repair (17), raising the possibility of alternative Chk1 inhibitor–based approaches in various malignancies.

To date, little information is available concerning the functional relationship between Chk1 and STAT3, although in nontransformed cells, Chk1 has been implicated in STAT3 activation following ischemic injury (23). However, the relationship between Chk1 and STAT3 activation in neoplastic cells in general, and in multiple myeloma cells in particular, remains largely unexplored. To address this question, we performed gene expression profiling of multiple myeloma cells exposed to established Chk1 inhibitors. These studies revealed that Chk1 inhibitors, administered at very low (e.g., nmol/L) concentrations, sharply attenuated the STAT3 gene expression profile. Moreover, using both pharmacologic and genetic approaches, we demonstrate that Chk1 inhibition markedly diminishes STAT3 Tyr₇₀₅ phosphorylation and DNA-binding activity accompanied by downregulation of STAT3-dependent genes (e.g., MCL-1, BCL-X_L, c-Myc), events shown to contribute functionally to Chk1 inhibitor lethality. Significantly, STAT3 inactivation by Chk1 inhibition was observed in the presence of IL6 or stromal cell factors. Collectively, these findings highlight a heretofore unrecognized link between the Chk1 and STAT3 pathways in multiple myeloma, and identify Chk1 inhibitors as novel and potent candidate STAT3 antagonists.

Human multiple myeloma cell lines U266, RPMI8226, OPM2, MM1.S, NCI-H929, KMS28BM, KMS28PE, ANBL-6 (1 ng/mL IL6), and KAS-6/1 (1 ng/mL IL6) cells were maintained in RPMI1640 supplemented with 10% FBS and penicillin-streptomycin. Bortezomib-resistant cells, U266/PS-R was established and maintained as described previously (24). Human marrow stroma cells (HS-5) were cultured in 10% FBS and penicillin-streptomycin.

For information regarding PF-477736 (PF), CEP-3891 (CEP), prexasertib (LY2606368, LY), Cryptotanshinone (Cry), BBI-608 (BBI), TTI-101 (TTI), Stattic, atovaquone, pervanadate (PV), ruxolitinib, recombinant Human IL6, and plasmids (see Supplementary Table S1).

Total RNA was separated by RNeasy Mini Kit then converted to cDNA using an RT² First Strand Kit (Qiagen) according to the manufacturer's instructions. Gene expression profiling was conducted using the Human Signal Transduction PathwayFinder RT² Profiler PCR Array (catalog no. PAHS-014Z, Qiagen, SABiosciences). Quantitative RT-PCR was conducted using an ABI 7900HT Real-time PCR Instrument (Applied Biosystems) following the array manufacturer's instructions. Relative gene expression was determined using the ΔΔC_T method. Data were generated using the Qiagen data analysis website.

Surface plasmon resonance (SPR) binding experiments were carried out at 25°C on a Reichert Technologies SR7500DC two-channel system equipped with CM5 chip using D-PBS with 0.05% Tween-20 and 5 μmol/L ATP as the running buffer. Chk1 protein (MW∼59,000, SignalChem, catalog No.C47-10H-100) was injected onto the surface on the left channel and captured to a level of 1727 μRIU, whereas the right channel was to serve as a reference for nonspecific binding of STAT3. Under a flow rate of 25 μL/minute, STAT3 protein (MW∼95,000, SignalChem, catalog no. S54-54BH) was injected over both channels at concentrations of 0.1, 0.2, 0.45, 0.9, and 2 μmol/L, respectively, for 6 minutes followed by a 6-minute dissociation in running buffer. The kinetic data were analyzed using TraceDrawer (Ridgeview Instruments) with a one to two model, while the equilibrium data were fitted using Scrubber (Biologic Software) to a one to two binding model.

Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, and permeabilized by incubation in 90% methanol on ice for 30 minutes. Cells were incubated with primary antibodies for 1 hour at room temperature: 1:200 anti-phospho-STAT3(Tyr705; Cell Signaling Technology, catalog no. 9145, RRID:AB_2491009), 1:400 anti-phospho-STAT3 (Ser727; Novus catalog no. IC4934U), 1:400 anti-STAT3 (Santa Cruz Biotechnology, catalog no. sc-8019, RRID:AB_628293), 1:400 anti-Chk1 (GeneTex, catalog no. GTX83385, RRID:AB_11175647), 1:10 CD-138-PE (Miltenyi Biotec, catalog no. 130-117-395); and secondary antibodies at room temperature, in the dark for 30 minutes: 1:1,000 Alexa Fluor 488 (Life Technologies, catalog no. A11070) and Alexa Fluor 647 (Life Technologies, catalog no. A21235); 1: 500 APC (Invitrogen, catalog no. 31984). Cells were also stained with DAPI 1:100 for 5 minutes before imaging. The cells were washed in 1,000 μL of PBS-2%FBS and recovered by centrifugation at 300 g for 5 minutes; and the incubation buffer for the antibodies was made from 5% BSA and 0.3% TritonX dissolved in PBS. Cells were resuspended in 60 μL of PBS-2%FBS and analyzed with an ImageStream (Amnis) image flow cytometer.

Kinase assays were performed using recombinant full-length human GST-STAT3 (SignalChem, C47-10H) together with recombinant full-length human His-Chk1 (SignalChem, S54-54G), in kinase buffer (20 mmol/L Hepes pH 7.4, 10 mmol/L MgCl₂, 10 mmol/L MgCl₂, 1 mmol/L EGTA, 0.1 mmol/L Na₃VO₄, 0.5 mmol/L NaF, 50 mmol/L β-glycerophosphatase, 50 mmol/L DTT) in the presence of 50 μmol/L ATP and 5 μCi [³²P]-ATP (EasyTide). The kinase reaction proceeded for 45 minutes at 30°C, and was stopped by the addition of NuPAGE LDS Sample Buffer (4X; Invitrogen NP0007), boiled for 10 minutes at 85°C, and analyzed by electrophoresis on 4%–12% SDS-polyacrylamide gel. Gels were stained with Coomassie Brilliant Blue (CBB), then fixed, dried, and subjected to autoradiography. A non-radioactive kinase assay was performed without cold ATP. Kinase products were analyzed by electrophoresis on 4%–12% SDS-polyacrylamide gel and transferred to polyvinylidene difluoride membranes, which were further stained Ponceau S. Immunoblotting was analyzed with rabbit anti-phospho-STAT3 (Tyr705, Cell Signaling Technology, catalog no. 2577), rabbit anti-phospho-STAT3 (Ser727, Cell Signaling Technology, catalog no. 9134), and rabbit anti-phospho-Chk1 (Ser296, Cell Signaling Technology, catalog no. 2349). Images were quantified and analyzed using ImageJ software (ImageJ, RRID:SCR_003070).

All studies were obtained with written informed consent from patients with multiple myeloma undergoing routine diagnostic aspirations. These studies were conducted in accordance with recognized ethical guidelines (e.g., the Declaration of Helsinki), and were approved by the Virginia Commonwealth Institutional Review Board (#MCC-8712-3A; MCC-02447; MCC-03340).

For multiple myeloma primary cell analysis, mononuclear cells were isolated from multiple myeloma patient bone marrows using the Ficoll Histopaque method (#10771, Sigma-Aldrich). CD138⁺ and CD138⁻ cells were separated using an MS⁺/LS⁺ column and a magnetic separator according to the manufacturer's instructions (Miltenyi Biotech).

Values represent the means ±SD for three separate experiments. The significance of differences between experimental variables was determined using the Student t test. Values were considered statistically significant at *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

All animal studies were Institutional Animal Care and Use Committee approved and performed in accordance with AAALAC, USDA, and PHS guidelines. NOD-SCID IL2Rgamma^null mice (Jackson Laboratories) were subcutaneously injected with 5 × 10⁶ U266 cells into the flank. The Chk1 inhibitor PF-477736 (Abmole, M1764) was prepared in a 1:1 (v/v) mixture of PBS and DMSO at a concentration of 10.0 mg/mL. When tumors grew to 300 mm³, PF (15 mg/kg per day) was administrated (intraperitoneally) for 3 days. Control animals received equal volumes of vehicle. Tumors were collected to perform immunoblotting and IHC staining.

For additional information and methods, see Supplementary Materials and Methods, and Supplementary Table S1.

In view of emerging evidence of non-canonical roles for Chk1 (25), gene expression profiling was performed on U266 multiple myeloma cells exposed (16 hours) to a low (100 nmol/L) concentration of the Chk1 inhibitor PF-477736 (Fig. 1A; ref. 26). These cells were selected because of their relatively high basal STAT3 activation and pronounced sensitivity to the pro-apoptotic effects of PF (Supplementary Fig. S1A, top, and Supplementary Fig. S1B). Lower basal STAT3 activation was observed in MM1.S, H929, 8226, and OPM2 cells, which was sharply increased by HS-5 conditioned medium (CM; Supplementary Fig. S1A, bottom). Marked reductions (e.g., > 2-fold) in gene expression profiling of multiple STAT3 pathway genes (e.g., MCL-1, BCL-X_L, c-Myc) were observed in U266 cells exposed to PF (Fig. 1A), results confirmed by quantitative PCR array at both short, for example, 4 hours and longer, for example, 16-hour intervals (Fig. 1B). Parallel reductions in mRNA expression were also observed in IL6-dependent KAS-6/1 cells exposed (2 hours) to PF or the Chk1 inhibitor CEP3891 (ref. 27; Supplementary Fig. S1C–S1E). These results indicate that pharmacologic Chk1 inhibitors repress the transcription of STAT3-dependent genes in multiple myeloma cells.

Western blot analysis revealed dose-dependent downregulation of STAT3 Y705 (but not S727) phosphorylation in U266 cells exposed to PF (Fig. 1C) or the Chk1 inhibitor LY2603618 (Fig. 1D). More detailed time course studies demonstrated prolonged Y705 dephosphorylation in U266 cells by PF, for example, up to 48 hours (Supplementary Fig. S1F). Notably, comparison studies demonstrated that low (e.g., 100–200 nmol/L) concentrations of PF or CEP were as or more potent than 1-log (Cry, BBI, TTI, Stattic) or 2-log (atovaquone) higher concentrations of other known STAT3 inhibitors (Fig. 1E). Finally, ImageStream flow cytometry demonstrated a significant reduction of DAPI and p-Y705 nuclear co-staining in PF-treated U266 cells (Fig. 1F). Similar results were obtained in U266 cells treated with CEP (Supplementary Fig. S1G). These findings indicate that Chk1 inhibitors block STAT3-Y705 nuclear translocation in multiple myeloma cells.

Attempts were then made to replicate these results in other cell types, including those displaying relatively low basal p-Y705 STAT3 activation stimulated by IL6 or HS-5 (human stroma cell) CM. Exposure of IL6-dependent KAS-6/1 cells to very low concentrations of PF or CEP (50–300 nmol/L) robustly reduced STAT3 Y705 phosphorylation by Western blot analysis but had minimal effects on STAT3 S727 (Fig. 2A and B). As in the case of U266 cells, PF and CEP were 1–2 logs more potent than other STAT3 inhibitors, for example, Cry 2.5 μmol/L (28) or atovaquone in diminishing Y705 phosphorylation (Fig. 2B) in KAS-6/1 as well as in bortezomib-resistant U266 cells (PS-R; Fig. 2C; ref. 24). Analogously, culture of OPM2 cells with CM or IL6 sharply increased STAT3 Y705 phosphorylation but had lesser effects on S727 (Fig. 2D and E). Coadministration of PF diminished both CM- and IL6-mediated STAT3 Y705 phosphorylation in these cells. Finally, ImageStream analysis confirmed the ability of PF and LY to block Y705 nuclear expression in CM- or IL6-stimulated OPM-2 cells (Fig. 2F).

Subsequent studies were undertaken to characterize further events involved in Chk1 inhibitor–mediated inactivation of STAT3. Consistent with its capacity to antagonize STAT3 Y705 phosphorylation, PF clearly diminished STAT3 dimerization in U266 cells at both 3 and 6 hours in a concentration-dependent manner (Fig. 3A). Electrophoretic mobility shift assays (EMSA) demonstrated that PF reduced STAT3-DNA binding in these cells at 6 and 16 hours (Fig. 3B). In addition, PF, LY, and CEP significantly reduced STAT3 binding activity at 6 or 16 hours (Fig. 3C). In parallel, both PF and CEP significantly diminished STAT3 binding activity in CM-treated OPM2 cells (Supplementary Fig. S2A and S2B).

Based upon these findings, ChIP assays were performed to assess the effects of PF on binding of STAT3 to the promoters of STAT3 downstream target genes. Treatment of cells with PF (100 or 500 nmol/L) significantly reduced STAT3 binding to c-Myc, BCL-X_L, and MCL-1 promoters (Fig. 3D). Consistently, both PF (200 nmol/L) and CEP (500 nmol/L) significantly diminished c-Myc, BCL-X_L, and MCL-1 mRNA levels in U266 cells (Fig. 3E), as well as in IL6-dependent KAS-6/1 cells (Supplementary Fig. S2C).

Finally, STAT3 promoter-reporter transcriptional activity was investigated in a 293t cell reporter system. Exposure of cells to IL6 sharply increased STAT3 transcriptional activity, and this was dramatically blocked by very low concentrations of PF (e.g., 50 nmol/L), which was more effective than 2-log higher concentrations of atovaquone (Supplementary Fig. S2D). Similar results were seen with CEP and LY (Supplementary Fig. S2E and S2F).

Attempts were then made to investigate the mechanism(s) by which Chk1 inhibition disrupted STAT3 signaling. To assess possible physical interactions between Chk1 and STAT3, SPR utilizing recombinant STAT3 and Chk1 was employed. As shown in Fig. 4A, a concentration-dependent increase in binding of STAT3 to Chk1 was observed. Furthermore, immunoprecipitation analysis revealed that administration of PF (200 nmol/L; 16 hours) to U266 cells reduced the amount of total STAT3 co-immunoprecipitating with Chk1 in both nuclear and cytoplasmic fractions, and of p-Y705 STAT3 associating with Chk1 in the nuclear fraction (Fig. 4B), raising the possibility that direct interactions may be involved in the Chk1/STAT3 nexus.

To explore the possible role of phosphatases in inhibition of STAT3 Y705 phosphorylation by Chk1 inhibitors, U266 cells were exposed (6 or 24 hours) to PF in the presence or absence of the specific phosphatase inhibitor PV. Co-exposure to PV antagonized PF-induced p-Y705 downregulation, particularly at 24 hours, but had little effect on p-S727 expression (Fig. 4C), raising the possibility that phosphatase induction may contribute to this interaction. However, PF diminished rather than induced Y580 phosphorylation of the tyrosine phosphatase SHP2 (Supplementary Fig. S3A). In contrast to a recent report involving a putative STAT3 inhibitor (29), PF reduced rather than increased expression of the PRL3 phosphatase, and had little effect on the SOCS3 phosphatase (Supplementary Fig. S3B). Furthermore, unlike atovaquone (11), PF failed to reduce GP130 or IL6R expression (Supplementary Fig. S3C).

An in vitro kinase assay was performed to determine whether PF might directly inhibit STAT3 Y705 phosphorylation. Interestingly, addition of His-Chk1 (0.1–0.5 μg) to GST-STAT3 in the presence of ³²P increased labeled phospho-STAT3 in a concentration-dependent manner (Fig. 4D). This event was attenuated, also in a concentration-dependent fashion, by co-administration of PF, which diminished p-Chk1 signal (Fig. 4E). Western blot analysis confirmed reductions in STAT3 Y705 (but not S727) phosphorylation in the presence of increasing concentrations of PF, along with downregulation of p-Chk1 S296 (Fig. 4F), consistent with effects on Chk1 downstream targets, (30), for example, downregulation of phospho-CDC25A and upregulation of Cyclin E (Supplementary Fig. S4A and S4B). These findings raise the possibility that Chk1 participates in STAT3 Y705 phosphorylation, and that this process may be directly disrupted by Chk1 inhibition.

Finally, in immunoprecipitation studies, the JAK kinase inhibitor ruxolitinib robustly blocked JAK2 Y570 phosphorylation, whereas PF had no effect (Fig. 4G, left). Both agents blocked STAT3 Y705 but not S727 phosphorylation. Consistent with these results, ruxolitinib but not PF abrogated JAK2 kinase activity in a cell-free assay (Fig. 4G, right). Similarly, JAK1, JAK3, and TYK2 were not dephosphorylated by PF (Supplementary Fig. S4C and S4D). These findings argue that PF inhibits STAT3 Y705 activation through a JAK/TYK-independent process.

To confirm the functional contribution of Chk1 to STAT3 activity, U266 cells inducibly expressing Chk1 short hairpin RNA (shRNA) were generated (Fig. 5A). Addition of doxycycline-inducible shChk1 partially diminished Chk1 expression accompanied by downregulation of STAT3 p-Y705. In parallel, three stable Chk1 shRNA knockdown clones were generated (KD1-3) which displayed marked downregulation of STAT3 p-Y705 (Fig. 5B). Compatible results were obtained in Chk1 CRISPR knockdown cells (CP1 and CP2; Supplementary Fig. S5A). Notably, stable Chk1 knockdown cells exhibited highly significant reductions in STAT3 binding activity (Fig. 5C), a finding confirmed by EMSA (Fig. 5D). Significantly diminished STAT3 binding activity was also observed in CRISPR knockdown cells (Supplementary Fig. S5B). In addition, KD1, KD2, and KD3 cells displayed significantly reduced STAT3 binding to the c-Myc, MCL-1, and BCL-X_L promoters (Fig. 5E–G) or mRNA expression by RT-PCR (Fig. 5H) compared with controls. Finally, Chk1 shRNA or CRISPR knockdown cells exhibited significantly reduced STAT3 p-Y705 nuclear intensity compared to EV controls (Fig. 5I; Supplementary Fig. S5C). Together, these findings support a functional contribution of Chk1 in regulating STAT3 nuclear disposition and DNA-binding activity in multiple myeloma cells.

To assess the functional significance of diminished STAT3 activation on the survival of cells exposed to PF, U266 cells expressing constitutively active STAT3 (mutation of A662C and N664C; ref. 31) were generated. Such cells, designated CA01 and CA02, displayed significantly increased basal STAT3 binding activity compared with empty-vector controls (Fig. 6A). Notably, these cells were significantly less sensitive to PF-induced lethality compared with controls (Fig. 6B), and displayed markedly reduced PF-mediated PARP and caspase cleavage as well as DNA damage (γH2A.X; Fig. 6C). Parallel studies were performed utilizing cells transfected with dominant-negative STAT3 [Y705F mutation (32)], designated DN01 and DN02, which exhibited significantly reduced STAT3 binding activity compared with controls (Fig. 6D). In contrast to CA cells, DN cells were significantly more sensitive to PF-induced cell death (Fig. 6E), and displayed diminished STAT3 p-Y705 expression and PARP cleavage (Fig. 6F) compared with empty-vector controls. These findings indicate that STAT3 inactivation plays a significant functional role in the lethal consequences of Chk1 inhibition in multiple myeloma cells.

Western blot analysis was performed to assess the impact of Chk1 interruption on expression of STAT3 target proteins. Exposure (3 and particularly 6 hours) of U266 cells to nmol/L concentrations of PF diminished expression of c-Myc, MCL-1, and BCL-X_L proteins (Supplementary Fig. S6A). Similar results were obtained in IL6-dependent KAS-6/1 cells (Supplementary Fig. S6B), OPM2 cells exposed to IL6 (Supplementary Fig. S6C), OPM2 cells cultured in the presence of CM (Supplementary Fig. S6D), and bortezomib-resistant PS-R cells (Supplementary Fig. S6E). In addition, as observed in the case of mRNA levels (Fig. 1E), treatment of U266 cells (6 hours) with PF or CEP was as or more effective than 1–2 log higher concentrations of multiple known STAT3 inhibitors in diminishing expression of c-Myc, MCL-1, and BCL-X_L protein levels (Supplementary Fig. S6F). Of note, Chk1 shRNA knockdown cells induced similar reductions in expression of these proteins (Supplementary Fig. S7A). Furthermore, enforced expression of constitutively active STAT3 abrogated PF-mediated protein downregulation (Supplementary Fig. S7B). Finally, enforced expression of MCL-1, c-Myc, or BCL-X_L significantly diminished PF-induced cell death (Supplementary Fig. S7C–S7E), arguing that disruption of STAT3 downstream targets plays a functional role in Chk1 inhibitor–mediated lethality.

Finally, attempts were made to extend these findings to primary human specimens and in vivo models. Two CD138⁺-isolated samples displayed clear p-Y705 and p-S727 activation but not in the CD138⁻ cell population (Fig. 7A, top). Interestingly, treatment (24 hours) of a third CD138⁺-isolated specimen with 500 nmol/L PF diminished p-Y705 expression (Fig. 7A, bottom). ImageStream flow cytometry exhibited significantly reduced pSTAT3 705Y nuclear intensity compared with controls (Fig. 7B). More detailed analysis of the effects of Chk1 inhibitor treatment was performed on primary samples in which sufficient numbers of CD138⁺ cells were available for Western blot analysis. In three such samples, PF or CEP (24 hours) reduced or abrogated p-Y705 expression. This was accompanied by variable reductions in the expression of c-Myc and MCL-1 protein (Fig. 7C).

Parallel studies were performed in NSG (NOD-SCID IL2Rgamma^null) mice inoculated in the flank with 5 × 10⁶ U266 cells and treated with PF (15 mg/kg, i.p., daily). Two mice were sacrificed on day 3, after which tumors were excised and proteins subjected to Western blot analysis. As shown in Fig. 7D, tumors obtained from PF-treated mice exhibited downregulation of p-Y705 accompanied by moderate reductions in the expression of c-Myc and MCL-1 proteins. IHC analysis confirmed the reduction in p-Y705–staining cells in tumors extracted from PF-treated mice (Fig. 7E; Supplementary Fig. S8). Furthermore, PF significantly suppressed tumor growth in vivo (P < 0.05; Supplementary Fig. S9A), with little evidence of toxicity (Supplementary Fig. S9B). Together, these findings suggest that inactivation of STAT3 by Chk1 inhibitors occurs in primary multiple myeloma cells and can be recapitulated in the in vivo setting.

The STAT3 transcription factor plays a key role in oncogenesis, transformation, metastasis, differentiation, and drug resistance, among multiple other activities (33). These characteristics have made it a high-value target in drug development, despite the fact that transcription factors are notoriously difficult to target therapeutically. Nevertheless, several candidate STAT3 inhibitors (e.g., TTI-101, BBI608) have now entered the clinic (NCT03195699, NCT03647839). In addition to the de novo development of STAT3 antagonists, attention has also focused on the ability of other classes of agents to disrupt STAT3 function. For example, CDK7 antagonists have been shown to interfere with STAT3 activity (34), as has the quinone antimicrobial atovaquone (11). The present results identify a heretofore unrecognized link between the Chk1 and STAT3 pathways that offers the potential for therapeutic intervention. Specifically, they demonstrate that a variety of clinically relevant Chk1 inhibitors potently disrupt STAT3 activation at extremely low (e.g., nmol/L) concentrations, both in vitro as well as in vivo. Significantly, the inhibitory effects of Chk1 inhibitors on STAT3 activation were observed at 1–2 logs lower than those reported for other STAT3 antagonists. While previous studies have suggested that STAT3 may regulate Chk1 responses in normal renal cells subjected to oxidative injury (23), this represents the first indication that Chk1 may operate upstream of and play a critical role in STAT3 activation in neoplastic cells.

The mechanism(s) by which Chk1 inhibition disrupts STAT3 function is (are) likely to be multi-factorial. STAT3 activation requires phosphorylation on Tyr705, which promotes dimerization and nuclear localization, necessary for DNA binding and activation (35). In contrast, phosphorylation on Ser727 is primarily responsible for enhancing transactivation (36). In the current studies, Chk1 inhibitors primarily blocked the former action, and had little effect on Ser727 phosphorylation. This resulted in diminished STAT3 dimerization, nuclear translocation, and activation of STAT3-dependent genes. While chemical and genetic approaches may exert disparate activities (37), it is noteworthy that Chk1 knockdown recapitulated the effects of Chk1 inhibitors on STAT3 function, arguing for on-target actions. The question of how Chk1 inhibition opposes STAT3 Tyr705 phosphorylation is clearly important to address. In this regard, activation of STAT3 may be mediated by diverse events, including cytokine-mediated stimulation through JAK2 or diverse alternative upstream kinases (e.g., Src or MEK1/2; ref. 38). However, in contrast to atovaquone, Chk1 inhibition failed to downregulate expression of the scaffolding protein GP130, implicated in STAT3 activation events (39). In addition, Chk1 inhibition had no discernible effects on JAK1, JAK2, JAK3, and TYK2 activation, arguing against the participation of these kinases in STAT3 modulation. Significantly, the results of the in vitro kinase assay suggested that PF may act directly to diminish STAT3 Tyr705 phosphorylation, raising the possibility that Chk1 represents a kinase responsible for STAT3 activation at this site. Although such a finding was unanticipated given the canonical view that Chk1 is a serine/threonine kinase (40), several lines of evidence argue strongly for this possibility. These include the findings that (i) multiple Chk1 inhibitors or (ii) Chk1 knockdown selectively blocked STAT3 Tyr705 but not Ser727 phosphorylation; (iii) results of the in vitro kinase assay demonstrating the capacity of recombinant Chk1 to promote STAT3 Tyr705 phosphorylation; and (iv) prediction of Tyr sites as potential targets of Chk1 action (Supplementary Table S2, http://www.cbs.dtu.dk/services/NetPhos/; http://sysbio.unl.edu/PhosphoSVM/prediction.php). Finally, the existence of an alternative STAT3-activating tyrosine kinase susceptible to Chk1 inhibitor actions cannot be completely excluded.

In addition to blocking STAT3 phosphorylation, Chk1 inhibitors might potentially act by increasing the expression/activity of phosphatases responsible for dephosphorylation. In this context, upregulation of the PRL phosphatase has recently been shown to promote IL6-mediated STAT3 activation in multiple myeloma cells through a GP130- and SHP2-dependent mechanism (29). However, in the current study, Chk1 inhibition reduced rather than increased PRL and SHP2 expression/activation, arguing against a role for these phosphatases in Chk1 inhibitor action. In addition, Chk1 inhibitors did not upregulate the SOCS3 phosphatase, also implicated in STAT3 regulation (29). On the other hand, the specific tyrosine phosphatase inhibitor PV substantially reduced the ability of PF to block STAT3 Tyr705 phosphorylation, raising the possibility that enhanced activity of yet to be defined phosphatases might contribute to STAT3 inactivation in this setting.

Previous reports have shown that STAT3 associates with Chk1 in non-neoplastic cells, for example, normal renal cells subjected to oxidative injury (23). Consistent with these findings, SPR analysis demonstrated a dose- and time-dependent association between Chk1 and STAT3. Moreover, co-administration of PF diminished the amount of STAT3 co-immunoprecipitating with Chk1 in both the cytoplasm and nucleus, although in the case of nuclear proteins, this could be attributed to disruption of STAT3 Y705 phosphorylation and nuclear transport. Consequently, while it is conceivable that direct Chk1/STAT3 interactions may be required for or contribute to STAT3 activation, the functional significance of this association and its disruption remain to be determined. Nevertheless, the possibility exists that in addition to antagonizing STAT3 phosphorylation, dimerization, and nuclear translocation, disruption of direct STAT3/Chk1 interactions by Chk1 inhibitors might contribute to STAT3 antagonism. Further studies will be necessary to confirm or refute this possibility.

Constitutive activation of STAT3 has been reported in 50% or more of patients with multiple myeloma (41). Representative of this population, multiple myeloma cells with high basal levels of phospho-STAT3 and presumably dependent upon this transcription factor such as U226 cells displayed robust inactivation in the presence of low Chk1 inhibitor concentrations. However, Chk1 inhibitors effectively inactivated STAT3 in cells with low basal STAT3 activation (e.g., OPM2) but stimulated with IL6 or stromal cell CM. In this regard, IL6-mediated STAT3 activation has been implicated in chemoresistance mediated by microenvironmental factors (4). Such findings raise the possibility that interrupting Chk1 may be an effective strategy not only in the subset of multiple myeloma cells displaying STAT3 addiction, but also in STAT3-independent cells protected from cell death by stromal cell factors. The connection between Chk1 and STAT3 may have specific relevance for multiple myeloma, a disease which has recently been shown to be particularly vulnerable to replicative stress (42). For example, both Chk1 and its immediate upstream effector ATR are prototypical inducers of replicative stress (42). Moreover, STAT3 modulates the DNA damage response pathway, in part by promoting DNA repair (43). In addition, STAT3 regulates the response to DNA damage through its pro-survival and pro-proliferation–related downstream targets, for example, MCL-1, BCL-X_L, and c-Myc (44). Thus, disruption of STAT3 signaling by Chk1 inhibitors may have dual effects on the DDR, for example, promotion of DNA damage as well as the resulting apoptotic response. In support of this notion, multiple myeloma cells expressing constitutively active STAT3 displayed diminished DNA damage (γH2A.X formation) and cell death in response to PF. Such a dual mechanism may be particularly relevant to cells such as multiple myeloma which are intrinsically susceptible to replicative stress.

Collectively, these findings may have implications for multiple myeloma and potentially other malignances dependent upon STAT3 for survival. Chk1 inhibitors have been evaluated in solid tumors, generally in combination with genotoxic agents, but relatively rarely in hematopoietic malignancies, and to the best of our knowledge, never in multiple myeloma. On the basis of the susceptibility of multiple myeloma to replicative stress, and the ability of Chk1 inhibitors to trigger this phenomenon (45), evaluation of such agents in multiple myeloma warrants attention. The same could be said of ATR inhibitors, which are also potent inducers of replicative stress (46). Another implication of these findings is that the subset of multiple myeloma cells that are dependent upon STAT3 for survival might be particularly vulnerable to Chk1 disruption due to (i) enhanced DNA damage and (ii) a lowered apoptotic threshold stemming from STAT3 inactivation. In addition, STAT3 inactivation could promote cell death even in STAT3-independent cells by blocking microenvironmental pro-survival signals mediated by IL-6 and STAT3. Finally, the current findings could provide mechanistic insights supporting possible combination strategies involving Chk1 inhibitors and STAT3. For example, we have previously described synergistic interactions between Chk1 and MEK1/2 inhibitors in multiple myeloma cells (47). However, whereas Chk1 inhibitors do not inactivate STAT3 Ser727, a process implicated in STAT3 transactivation (48), MEK1/2 is known to phosphorylate STAT3 Ser727 (49). It is possible that combined therapy may more profoundly inactivate STAT3 by triggering dephosphorylation at both sites, thereby promoting cell death. Accordingly, studies designed to test this concept, as well as the ability of clinically relevant ATR inhibitors to interact analogously in replication stress-susceptible multiple myeloma cells are currently underway.

S.M. Sebti reports grants from NIH and Leukemia and Lymphoma Society of America during the conduct of the study. S. Grant reports grants from NIH 1RO1CA205607 and LSA R6508-18 during the conduct of the study. No disclosures were reported by the other authors.

L. Zhou: Conceptualization, data curation, supervision, validation, investigation, visualization, methodology, writing–original draft, writing–review and editing. X. Pei: Resources, formal analysis, validation, investigation, methodology. Y. Zhang: Formal analysis, validation, methodology. Y. Ning: Formal analysis, validation, investigation, methodology. L. Li: Formal analysis, validation, investigation. X. Hu: Validation, investigation, methodology. S.L. Chalasani: Validation, investigation, methodology. K. Sharma: Validation, investigation, methodology. J. Nkwocha: Formal analysis, validation, investigation. J. Yu: Methodology. D. Bandyopadhyay: Resources, software, formal analysis. S.M. Sebti: Conceptualization, supervision. S. Grant: Conceptualization, supervision, funding acquisition, validation, writing–original draft, writing–review and editing.

This work was supported by awards 1R01CA205607-01 (S. Grant) and an award from the Leukemia and Lymphoma Society of America (R6508-18; S. Grant). Services in support of the research project were provided by the Virginia Commonwealth University Massey Cancer Center Tissue and Data Acquisition and Analysis Core, Mouse Models Core Laboratory, and Flow Cytometry Shared Resource, supported, in part, with funding from NIH-NCI Cancer Center Support Grant P30 CA016059.

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