Stimulation of the BCR activates JAK2 and STAT3 in CLL cells.
The JAK1/2 inhibitor ruxolitinib induces apoptosis of CLL cells.
In chronic lymphocytic leukemia (CLL), stimulation of the B-cell receptor (BCR) triggers survival signals. Because in various cells activation of the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway provides cells with survival advantage, we wondered whether BCR stimulation activates the JAK/STAT pathway in CLL cells. To stimulate the BCR we incubated CLL cells with anti-IgM antibodies. Anti-IgM antibodies induced transient tyrosine phosphorylation and nuclear localization of phosphorylated (p) STAT3. Immunoprecipitation studies revealed that anti-JAK2 antibodies coimmunoprecipitated pSTAT3 and pJAK2 in IgM-stimulated but not unstimulated CLL cells, suggesting that activation of the BCR induces activation of JAK2, which phosphorylates STAT3. Incubation of CLL cells with the JAK1/2 inhibitor ruxolitinib inhibited IgM-induced STAT3 phosphorylation and induced apoptosis of IgM-stimulated but not unstimulated CLL cells in a dose- and time-dependent manner. Whether ruxolitinib treatment would benefit patients with CLL remains to be determined.
Chronic lymphocytic leukemia (CLL) cells traffic between the peripheral blood (PB) and lymphoid organs,1,2 in which they are amenable to extracellular signals that protect them from apoptosis and stimulate their proliferation.3 CLL cells obtained from lymph nodes expressed B-cell receptor (BCR) activation genes, suggesting that antigen stimulation of the BCR activates antiapoptotic signals.4,5
In circulating CLL cells, the signal transducer and activator of transcription 3 (STAT3) is constitutively phosphorylated on serine-727 residues.6,7 Tyrosine phosphorylated (p) STAT3 is rarely detected in unstimulated circulating CLL cells in PB. However, extracellular factors such as interleukin-6 (IL-6) induce transient tyrosine phosphorylation of STAT3 in CLL cells.7 Tyrosine pSTAT3 shuttles to the nucleus, binds to DNA, and activates transcription of antiapoptosis genes.7-11 Whether stimulation of the BCR induces tyrosine pSTAT3 as well is unknown. Because stimulation of normal BCRs induces tyrosine phosphorylation of STAT3,12 we sought to determine whether stimulation of CLL-cell BCRs induces tyrosine phosphorylation of STAT3 and which signaling pathway or pathways are engaged in this process.
PB cells were obtained from untreated CLL patients (supplemental Table 1; available on the Blood Web site) who were followed at the University of Texas MD Anderson Cancer Center Leukemia Center from 2011 to 2013 after the patients gave Institutional Review Board–approved informed consent to participate in the study. The study was conducted in accordance with the Declaration of Helsinki. The cells were fractionated using Histopaque-1077 (Sigma-Aldrich, St. Louis, MO).
Activation of the BCR
Freshly isolated CLL B cells were resuspended in a culture medium as described previously.7 BCR stimulation was performed via incubation with 10 μg/mL goat F(ab′)2 anti-human IgM (MP Biomedicals, Santa Ana, CA).
Western immunoblotting and immunoprecipitation
Western immunoblotting and immunoprecipitation studies were performed as described previously.7 The following primary antibodies were used: monoclonal mouse anti-human STAT3 (BD Biosciences, Palo Alto, CA); rabbit anti-human serine pSTAT3, rabbit anti-human tyrosine pSTAT3, rabbit anti-human Janus kinase 2 (JAK2), and rabbit anti-human tyrosine pJAK2 (Cell Signaling Technology, Beverly, MA); mouse anti-human lamin B, mouse anti-human S6, and poly(adenosine 5′-diphosphate-ribose) polymerase (PARP; Calbiochem, Billerica, MA); and mouse anti-human β-actin (Sigma-Aldrich).
Isolation of nuclear and cytoplasmic extracts
Nondenatured nuclear and cytoplasmic extracts of CLL cells were prepared using an NE-PER extraction kit (Thermo Fisher Scientific, Rockford, IL) and confirmed western blot–based detection of the nuclear protein lamin B and cytoplasmic S6 ribosomal proteins.7
The rate of cellular apoptosis was analyzed via flow cytometry using double staining with a Cy5-conjugated annexin V and propidium iodide (PI; BD Biosciences) according to the manufacturer’s instructions.
Confocal microscopy was performed as previously described with 4,6-diamidino-2-phenylindole staining (Invitrogen, Carlsbad, CA), S6, and tyrosine pSTAT3 (BD Biosciences, San Diego, CA).7
Polymerase chain reaction (PCR)
RNA was isolated using an RNeasy purification procedure (Qiagen Inc., Valencia, CA). Five hundred nanograms of total RNA was used in 1-step quantitative reverse transcription–PCR (qRT-PCR; Applied Biosystems, Foster City, CA). Real-time PCR and qRT-PCR were performed as previously described.7
Results and discussion
To determine whether activation of the BCR in CLL cells induces tyrosine phosphorylation of STAT3, CLL cells from PB were incubated with anti-IgM antibodies, which are known to activate the BCR in CLL cells.13,14 In all experiments, anti-IgM antibodies induced tyrosine pSTAT3 and slightly increased serine pSTAT3 levels. Contrary to IL-6 that induced tyrosine pSTAT3 within 15 minutes (Figure 1A), anti-IgM antibodies induced phosphorylation of STAT3 within 2 hours (Figure 1B). However, the anti-IgM–induced phosphorylation of STAT3 was short lived. Two hours after IgM washout, tyrosine pSTAT3 was no longer detected (representative results from 3 identical separate experiments are depicted in Figure 1A-B).
Following cytokine-induced phosphorylation, STAT3 translocates to the nucleus.10 To determine whether BCR-induced tyrosine pSTAT3 also shuttles to the nucleus and activates STAT3-target genes, we prepared cytoplasmic and nuclear extracts of IgM-stimulated CLL cells and analyzed them using western immunoblotting.7 As shown in Figure 1C, tyrosine pSTAT3 was detected in the cytoplasmic and nuclear fractions of IgM-stimulated CLL cells. Similarly, confocal microscopy studies detected tyrosine pSTAT3 in the nucleus of IgM-stimulated, but not unstimulated, CLL cells (Figure 1D). RT-PCR revealed that anti-IgM antibodies upregulated STAT3-target genes whose levels were increased by 1.8-fold (BCL2) to 24-fold (Cyclin D1), as assessed by qRT-PCR (Figure 1E). Taken together, these results suggest that stimulation of the BCR induces tyrosine phosphorylation of STAT3 and mildly increases levels of serine pSTAT3, and that phosphorylation of STAT3 either at serine or tyrosine residues activates transcription.
To determine which signaling pathways are engaged in BCR-induced STAT3 phosphorylation, we incubated CLL cells from 2 patients with or without anti-IgM antibodies and assessed the exposure to 3 kinase inhibitors. As shown in Figure 2A, 1 µM of the Abl and Lyn kinase inhibitor dasatinib15 completely blocked IgM-mediated phosphorylation of Lyn kinase, whereas the levels of IgM-induced pSTAT3 remained unchanged, suggesting that BCR-mediated tyrosine phosphorylation of STAT3 is Lyn independent. Also, the mitogen-activated protein kinase signaling pathway inhibitor U0126 (50 µM) downregulated the expression of serine pSTAT3 as described previously16 but did not affect the levels of tyrosine pSTAT3. Conversely, the JAK1/2 inhibitor ruxolitinib17 markedly reduced the level of tyrosine but not serine pSTAT3 in IgM-stimulated CLL cells in a dose-dependent manner (Figure 2A-B), suggesting that activation of the BCR induces tyrosine phosphorylation of STAT3, likely via activation of JAK2.
To confirm that BCR stimulation activates the JAK2/STAT3 pathway in CLL cells, we incubated CLL cells from 4 patients with or without anti-IgM antibodies for 2 hours. Subsequently, we immunoprecipitated the cell lysates with anti-JAK2 antibodies. As shown in Figure 2C, we detected both pJAK2 and tyrosine pSTAT3 in the JAK2-immunoprecipitated lysates of cells incubated with but not without anti-IgM antibodies, suggesting that stimulation of the BCR induces JAK2 phosphorylation and that pJAK2 binds to and phosphorylates STAT3 on tyrosine-705 residues in CLL cells.
Because pSTAT3 provides CLL cells with a survival advantage7 and exposure to ruxolitinib inhibited tyrosine phosphorylation of STAT3 in IgM-stimulated CLL cells, we investigated the effect of exposure to ruxolitinib on CLL-cell viability. As shown in Figure 2D-E, ruxolitinib, but not dasatinib or U0126, induced apoptosis of IgM-stimulated CLL cells in a dose- and time-dependent manner. This effect was observed in IgM-stimulated but not in unstimulated CLL cells (Figure 2E-F).
The recently described tonic low-grade activation of the BCR18 does not induce tyrosine phosphorylation of STAT3, for which full-scale BCR stimulation resulting in activation of JAK2 is required. Whereas stimulation of the BCR induces rapid Syk or extracellular signal-regulated kinase 1/2 phosphorylation,19 stimulation of the BCR for at least 2 hours was needed to induce tyrosine pSTAT3, suggesting that activation of transcription is required, a slow signaling pathway(s) is recruited, or both. Conversely, BCR-induced tyrosine phosphorylation of STAT3 is short lived and therefore rarely detected in circulating CLL cells. In vitro models1 and gene expression profiles of CLL cells in PB and lymph nodes5 agree with these findings. Upon migration to PB, CLL cells are no longer stimulated by their microenvironment. Once the BCR is no longer engaged, the gene signature associated with BCR activation changes drastically.7
Taken together, our findings suggest that stimulation of the BCR activates the JAK2/STAT3 pathway in CLL cells. Whether treatment with ruxolitinib is clinically beneficial in patients with CLL remains to be determined.
The online version of this article contains a data supplement.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 USC section 1734.
We thank Susan Smith for obtaining the patients’ clinical data and Don Norwood for editing the manuscript.
This work was supported in part by the National Institutes of Health through MD Anderson Cancer Center Support Grant CA016672 and the CLL Global Research Foundation.
Contribution: M.J.K., Z.E., and S.V. were responsible for conception and design of the study; J.A.B., A.F., M.J.K., S.O., and W.G.W. were responsible for provision of study materials or patients; J.Y.W. performed the western blot and IP experiments; D.M.H. performed the PI/annexin assay; P.L. helped in the IP experiments; Z.L. performed the western blot and IP experiments; I.H.-H. helped in the IP and western blot experiments; U.R. and Z.E. wrote the manuscript; and U.R., J.Y.W., D.M.H., Z.L., P.L., I.H.-H., A.F., J.A.B., S.O., N.J., S.V., W.G.W., M.J.K., and Z.E. approved the final manuscript.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Zeev Estrov, Department of Leukemia, Unit 428, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030; e-mail: firstname.lastname@example.org.