Constitutive NF-κB Activation Confers Interleukin 6 (IL6) Independence and Resistance to Dexamethasone and Janus Kinase Inhibitor INCB018424 in Murine Plasmacytoma Cells*

Myeloma cells are dependent on IL6 for their survival and proliferation during the early stages of disease, and independence from IL6 is associated with disease progression. The role of the NF-κB pathway in the IL6-independent growth of myeloma cells has not been studied. Because human herpesvirus 8-encoded K13 selectively activates the NF-κB pathway, we have used it as a molecular tool to examine the ability of the NF-κB pathway to confer IL6 independence on murine plasmacytomas. We demonstrated that ectopic expression of K13, but not its NF-κB-defective mutant or a structural homolog, protected plasmacytomas against IL6 withdrawal-induced apoptosis and resulted in emergence of IL6-independent clones that could proliferate long-term in vitro in the absence of IL6 and form abdominal plasmacytomas with visceral involvement when injected intraperitoneally into syngeneic mice. These IL6-independent clones were dependent on NF-κB activity for their survival and proliferation but were resistant to dexamethasone and INCB018424, a selective Janus kinase 1/2 inhibitor. Ectopic expression of human T cell leukemia virus 1-encoded Tax protein, which resembles K13 in inducing constitutive NF-κB activation, similarly protected plasmacytoma cells against IL6 withdrawal-induced apoptosis. Although K13 is known to up-regulate IL6 gene expression, its protective effect was not due to induction of endogenous IL6 production but instead was associated with sustained expression of several antiapoptotic members of the Bcl2 family upon IL6 withdrawal. Collectively, these results demonstrate that NF-κB activation cannot only promote the emergence of IL6 independence during myeloma progression but can also confer resistance to dexamethasone and INCB018424.

Several such proteins, such as the SV40 large and small T antigens and the human papillomavirus E6 and E7 proteins, have been successfully used as molecular tools to discern the role of cellular signaling pathways in various biological processes (18). The human herpesvirus 8 (HHV8, also known as Kaposi's sarcoma-associated herpesvirus)-encoded K13 protein contains two tandem death effector domains that are also present in the prodomain of caspase 8/FLICE. Proteins with two death effector domains are also found in several other viruses and include MC159L and MC160L from the Molluscum contagiosum virus and E8 from equine herpesvirus 2 (EHV2) (19 -21). These proteins were originally believed to protect virally infected cells from death receptor-induced apoptosis by blocking the recruitment and/or activation of caspase 8/FLICE and as such were collectively referred to as viral FLICE inhibitory proteins or vFLIPs (19 -21). However, subsequent work by our laboratory and others showed that K13 does not act as a vFLIP but instead directly interacts with the NEMO/IKK␥ subunit of the IKK complex to selectively activate the NF-B pathway (22)(23)(24). In this study, we have taken advantage of this unique ability of K13 to selectively activate the NF-B pathway and used it as a molecular tool to study the role of the NF-B pathway in IL6independent growth of murine plasmacytoma cells.
Retrovirus, Lentivirus Constructs, and Virus Infection-Retrovirus constructs containing C-terminal FLAG epitopetagged wild-type and mutant vFLIP K13 and E8 were generated in murine stem cell virus (MSCV) neo-based retroviral vector, and amphotropic viruses were generated and used for infection as described previously (23). Lentivirus constructs encoding C-terminal FLAG-tagged wild-type Tax and its mutants (M22 and M47) were generated in pLENTI6/V5-based vector (Invitrogen). A retroviral vector expressing the firefly luciferase gene was constructed in the pRetroQ-RSV vector (Clontech) in which the CMV promoter had been replaced with an RSV promoter. The MSCV-Bcl-2-IRES-GFP and MSCV-Bcl-xL-IRES-GFP constructs were kindly provided by Dr. Emily Cheng (Human Oncology and Pathogenesis Program, Memorial Sloan-Kettering Cancer Center, New York, NY). The MSCVpuro-WT-Mcl-1 construct was a kind gift from Dr. Opferman (Department of Biochemistry, St. Jude Children's Research Hospital, Memphis, TN). Recombinant retroviruses and lentiviruses were generated in the HEK293-FT cells as described previously and used to infect T1165 and B9 cells (25,26). All infections were carried out in the presence of 8 g/ml polybrene (Sigma). Post-infection, cells were cultured in normal growth media containing the appropriate drugs to select positive clones or sorted based on GFP fluorescence.
Cell Viability and Cell Cycle Assays-Cells from exponentially growing cultures were washed three times with human IL6 free medium and plated in an untreated flat-bottom 96-well plate at a density of 5 ϫ 10 3 cells/well in the presence or absence of hIL6. Cell viability was measured after 48 h using the MTS reagent (3-4,5-dimethylthiazol-2yl)-5-(3-carboxy-methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) following manufacturer's instructions (Promega, Madison, WI). Absorbance of viable cells was measured at 490 nm with 600 nm as a reference wavelength. Percent cell survival was calculated based on the reading of cells grown in the presence of hIL6 as 100%. DNA content analysis was performed as described previously (25).
ELISA for Murine IL6-Cells were harvested by centrifugation, washed three times in medium lacking growth factors, and then set up at 10 5 cells/ml in IL6-free medium for 72 h. Cells were centrifuged, and the supernatant was filtered and assayed for mIL6 using an IL6 ELISA kit (eBioscience) following the recommendations of the manufacturer.
Assays for Nuclear NF-B DNA-binding Activity-Nuclear proteins were extracted and used for measuring the status of NF-B DNA binding by EMSA or an ELISA-based transfector kit (Clontech), as described previously (22,27).
Animal Experiments-T1165 vector and T1165-K13 ϪIL6 cells were transduced with a pRetroQ-RSV-Luc retroviral vector that expresses the firefly gene under an RSV promoter, and infected cells were selected with puromycin. Subsequently, 4to 6-week-old BALB/cAnNCr mice (Charles River Laboratories, Wilmington, MA) were inoculated intraperitoneally with 10 ϫ 10 7 cells. Tumor growth in the peritoneum was monitored weekly by physical examination and bioluminescence imaging for 10 weeks. For bioluminescence imaging, after mild anesthesia with isoflurane, animals were injected intraperitoneally with an aqueous solution of D-luciferin (Biosynth, Naperville, IL) at 150 g/g body weight in PBS, and firefly luciferase activity was deter-mined using the IVIS200 system (Caliper, Hopkinton, MA). At autopsy, cells were harvested from an abdominal plasmacytoma, and the spleen of a mouse was injected with T1165-Luc-K13 ϪIL6 cells and cultured in RPMI medium with 10% FCS in the absence of IL6. In parallel, cells were harvested from the spleen of an animal injected with the T1165-Luc-vector cells. After ϳ1 week, clumps of proliferating cells were clearly visible in the cultures established from the plasmacytoma and spleen of the T1165-Luc-K13 ϪIL6injected animal but were absent in those established from the spleen of the T1165-Luc vector-injected animal. Cell lysates were prepared and used for immunoblotting to confirm the expression of K13 as described above. All animal procedures were conducted according to an Institutional Animal Care and Use Committee-approved protocol in the University of Pittsburgh Cancer Center animal facility.
Statistical Analyses-Two-tailed paired Student's t test was used to test for differences between two groups. Differences with a p Յ 0.05 were considered as statistically significant. All experiments were repeated a minimum of three times with triplicate samples.

K13 Protects Mouse T1165 Plasmacytoma Cells against IL6
Withdrawal-induced Apoptosis-The murine T1165 plasmacytoma cell line requires IL6 for survival and proliferation (28). We used retroviral gene transfer to generate polyclonal populations of these cells expressing K13 or an empty vector. The expression of K13 protein was confirmed by immunoblotting ( Fig. 1A), and its ability to protect against IL6 withdrawal-induced apoptosis was examined by growing the T1165-K13 and -vector cells in the presence or absence of IL6. As shown in Fig.  1B, the T1165-vector cells underwent a dramatic reduction in cell viability within 24 -48 h upon withdrawal from IL6, whereas the T1165-K13 cells were remarkably resistant (Fig.  1B). The lack of apoptosis in the T1165-K13 cells was confirmed by staining with SYTOX Green, a membrane-impermeable nuclear dye (Fig. 1C). While IL6 withdrawal from T1165vector cells resulted in the appearance of cells with brightly stained, condensed, and fragmented nuclei suggestive of apoptosis, they were absent among K13-expressing cells (Fig. 1C). Moreover, a DNA content analysis revealed that withdrawal of T1165-vector cells from IL6 resulted in appearance of cells with sub-G 0 /G 1 DNA content, which were not seen among the T1165-K13 cells (Fig. 1D). Taken collectively, these results demonstrate that K13 protects T1165 plasmacytoma cells against IL6 withdrawal-induced apoptosis.
Protective Effect of K13 against IL6 Withdrawal-induced Apoptosis Is Associated with Constitutive NF-B Activation-Continuous culture of K13-expressing T1165 cells in the absence of IL6 readily generated cells that were completely independent of IL6 and could proliferate long-term in IL6-free medium. These IL6-independent cells were designated T1165-K13 ϪIL6 . K13 activates NF-B by inducing phosphorylation of IB␣, which results in its proteasomal-mediated degradation, allowing the released NF-B subunits to enter the nucleus and bind to the promoters of its target genes (23). To examine the role of NF-B pathway in IL6-independent growth of the T1165-K13 ϪIL6 cells, we performed an electrophoretic mobility shift assay. As shown in Fig. 2A, this assay revealed a marked increase in the NF-B DNA-binding activity in the nuclear extracts of the T1165-K13 ϪIL6 cells as compared with the T1165-vector cells. Consistent with the above results, immunoblot analysis showed constitutive phosphorylation of IB␣ and loss of total IB␣ expression in the T1165-K13 ϪIL6 cells (Fig.  2B). However, there was no significant increase in the phosphorylation of JNK and Akt in the T1165-K13 ϪIL6 cells (Fig. 2C). In fact, consistent with the known ability of IL6 to activate the Akt pathway (29), the phosphorylation of Akt was slightly reduced in the T1165-K13 ϪIL6 cells, which were grown in IL6-free medium. Collectively, these results confirmed our previous report that K13 selectively activates the NF-B pathway (30). The involvement of the NF-B pathway in the protective effect conferred by K13 was further supported by generation of T1165 cells expressing an NF-B-defective mutant of K13 (K13-58AAA) (31). Unlike T1165-K13 cells, T1165-K13-58AAA showed no protection against IL6 withdrawal-induced apoptosis (Fig. 2D). Similarly, expression of equine herpesvirus vFLIP E8, a structural homolog of K13 that lacks the ability to activate NF-B (22), failed to protect T1165 cells against IL6 withdrawal-induced apoptosis (Fig. 2D). Thus, the protective effect of K13 against IL6 withdrawal-induced apoptosis is associated with NF-B activation.
Protective Effect of K13 against IL6 Withdrawal-induced Apoptosis Is Reversed by Bay-11-7082-To confirm the involvement of NF-B activation in the protective effect of K13 against IL6 withdrawal-induced apoptosis, we took advantage of Bay-11-7082, a specific inhibitor of NF-B that is known to block K13-induced NF-B activation (32). Treatment with up to 1 M Bay-11-7082 had no significant effect on the survival of T1165vector cells (Fig. 3A). In contrast, T1165-K13 ϪIL6 cells were highly sensitive to this compound and underwent substantial cell death at a concentration of as low as 0.25 M (Fig. 3A). In addition, T1165-K13 ϪIL6 cells demonstrated preferential sen-  sitivity to arsenic trioxide, another known inhibitor of K13induced NF-B (Fig. 3B) (33). However, T1165-K13 ϪIL6 cells were relatively resistant to cell death induced by dexamethasone (Fig. 3C), a drug commonly used for the treatment of plasma cell neoplasms. Collectively, the above studies demonstrate that the NF-B activity cannot only promote the emergence of IL6-independent plasmacytoma cells but can also confer on them resistance to dexamethasone.
Tax-induced NF-B Activation Confers IL6 Independence on plasmacytoma Cell Lines-The human T-cell leukemia virus-1 (HTLV-1)-encoded Tax protein resembles K13 in constitutively activating the NF-B pathway by interacting with NEMO (34). As an independent confirmation of the involvement of the NF-B pathway in the protective effect of K13 against IL6 withdrawal-induced apoptosis, we generated stable populations of T1165 cells expressing wild-type Tax and its two mutants, M22 and M47, respectively (Fig. 4A). The M22 mutant is known to lack the ability to activate NF-B, whereas the M47 mutant is inactive in the cAMP response element-binding protein/activating transcription factor-1 pathway but retains NF-B activity (35). Accordingly we observed increased NF-B activity in T1165 cells expressing wild-type Tax and its M47 mutant but not in those expressing the M22 mutant (Fig. 4B). Consistent with the key role of the NF-B pathway in protection against IL6 withdrawal-induced cell death, we observed that T1165 cells expressing the wild-type Tax and its M47 mutant were protected from IL6 withdrawal-induced cell death, whereas no protection was observed in cells expressing the M22 mutant (Fig. 4C). Taken together, the above results demonstrate that constitutive activation of the NF-B pathway by viral proteins confers IL6 independence on IL6-dependent plasmacytoma cells.
Protective Effect of K13 against IL6 Withdrawal-induced Apoptosis Is Not Due to Stimulation of Endogenous IL6 Production-K13-induced NF-B has been shown to stimulate IL6 production (36). Therefore, we tested the hypothesis that the protective effect of K13 against IL6withdrawal-induced apoptosis is due to stimulation of endogenous IL6 production and autocrine/paracrine signaling. Surprisingly, an ELISA assay did not reveal the presence of IL6 in the supernatant of T1165-K13 cells (Fig. 5A). Similarly, there was no IL6 production in T1165 cells treated with 10 ng/ml TNF-␣ for 24 h (Fig. 5B). Furthermore, the conditioned medium collected from T1165-K13 cells failed to confer protection against IL6 withdrawalinduced cell death when added to a fresh batch of T1165 cells (Fig. 5C). Although the above studies demonstrated a lack of IL6 secretion in T1165-K13 cells, they did not rule out the possibility of intracellular IL6 signaling mediated by cytosolic interaction between IL6 and its receptor. IL6 exerts its intracellular effects through the JAK/STAT signaling pathway. As such, we examined the phosphorylation status of STAT1 and STAT3, two downstream mediators of IL6 signaling, in the T1165-vector and T1165-K13 cells grown in the absence or presence of IL6. Immunoblotting with p-STAT1 (Tyr-701) and p-STAT3 (Tyr-705) revealed significant phosphorylation of STAT1 and STAT3 in the T1165-vector and T1165-K13 cells grown in the presence of IL6 but not in its absence (Fig. 5D). INCB018424, a selective JAK1 and JAK2 inhibitor, is known to inhibit IL6 signaling (37). As an independent test of the lack of involvement of IL6 signaling in the survival of T1165 K13 ϪIL6 cells, we tested their resistance to INCB018424. As shown in Fig. 5E, T1165-K13 ϪIL6 cells demonstrated a marked resistance to this compound as compared with the T1165-vector cells. Taken collectively, the above results argue against the role of intracellular IL6-signaling in the survival of T1165-K13 ϪIL6 cells.

Protective Effect of K13 against IL6 Withdrawal-induced Apoptosis Is Associated with Block in Caspase Activation and Sustained Expression of Bcl2 Family Members upon IL6
Withdrawal-To understand the mechanism by which K13 protects T1165 cells against IL6 withdrawal-induced apoptosis, we examined the status of caspase 3 and Bcl2 family members. As shown in Fig. 5F, growth of T1165-vector cells in IL6-free medium for 12-18 h resulted in marked increase in the appearance of cleaved caspase 3, suggestive of caspase 3 activation. This was accompanied by cleavage of PARP, one of the downstream targets of caspase 3, and both of these events were significantly blocked in the K13-expressing cells (Fig. 5F).
To gain an understanding into the mechanism by which K13 expression blocks caspase 3 activation, we examined the status of several Bcl2 family members in the T1165-K13 and -vector cells that had been grown in the presence or absence of IL6. In the presence of IL6, T1165-K13 cells demonstrated an equivalent expression of Mcl-1, Bcl-2, and Bcl-xL as compared with the T1165-vector cells (Fig. 5F). However, although withdrawal of IL6 for 12-18 h resulted in a significant decline in the expres- sion of Mcl-1, Bcl-2, and Bcl-xL in the T1165-vector cells, the expression of these proteins was relatively well maintained in the T1165-K13 cells (Fig. 5F). Next, to examine whether ectopic expression of Bcl2 family members could suppress apoptosis upon IL6-withdrawal, we generated a stable populations of T1165 cells expressing Bcl-2, Bcl-xL, and Mcl-1 (Fig. 5G). These stable cells were significantly protected from IL6 withdrawal-induced cell death as compared with the empty vector-expressing cells (Fig. 5H). These results suggest that K13-induced NF-B protects against IL6 withdrawal-induced apoptosis by maintaining the expression of antiapoptotic members of the Bcl2 family.

K13 Protects B9 Plasmacytoma Cells against IL6 Withdrawal-induced
Apoptosis-To demonstrate that the protective effect of K13 against IL6 withdrawal-induced apoptosis is not limited to T1165 cells, we generated stable clones of IL6-dependent B9 plasmacytoma expressing K13 or an empty vector using retroviral gene transfer (Fig. 6A). Similar to the T1165-K13 cells, B9 cells expressing K13 were significantly protected against IL6 withdrawal-induced cell death as compared with the empty vector-expressing cells (Fig. 6B). Additionally, the protection conferred by K13 was reversed by NF-B inhibitor Bay-11-7082 and was not associated with phosphorylation of STAT1 and STAT3 (Fig. 6, C and D). Taken collectively with the studies using T1165 cells, the above results demonstrate that although K13 protects cells against IL6 withdrawal-induced apoptosis via NF-B activation, this effect is not mediated through NF-B-induced endogenous IL6 production.
Constitutive NF-B Activation Promotes Peritoneal Plasmacytomas without Pristane Conditioning-Murine plasmacytoma cells are not only dependent on IL6 for their in vitro growth but also require it for their growth in vivo (4). Thus, T1165 cells form peritoneal plasmacytoma only if the peritoneal cavity had been preconditioned with pristane, an inflammatory agent that induces chronic inflammation with copious IL6 production (4,38). To study the effect of constitutive NF-B activation on the ability of mouse plasmacytoma cells to grow in vivo in the absence of pristane conditioning, we used retroviral-mediated gene transfer to express the firefly luciferase (Luc) gene in the T1165 vector and T1165-K13 ϪIL6 cells. We injected 1 ϫ 10 7 T1165-Luc-vector and T1165-Luc-K13 ϪIL6 cells intraperitoneally into the syngeneic Balb/cAnNcr mice (n ϭ 9) and followed the development of plasmacytomas by weekly physical examination and bioluminescence imaging over the ensuing 1-3 months. The mice injected with the T1165-Luc-vector cells demonstrated no physical abnormalities. However, those injected with T1165-Luc-K13 ϪIL6 cells developed enlarged abdomens (Fig. 7A). Bioluminescence imaging confirmed intra-abdomen growth of tumors in all the T1165-Luc-K13 ϪIL6 -injected mice, whereas no tumor development was detected in the mice injected with T1165-Luc-vector cells (Fig. 7B). Autopsy not only confirmed the presence of plasmacytomas but also showed the enlargement of spleen and liver in the T1165-Luc-K13 ϪIL6 -injected mice (Fig. 7, C and D, and data not shown). Finally, T1165-Luc-K13 ϪIL6 cells were easily cultured from the spleen of these mice (Fig. 7E). Collectively, the above studies demonstrate that K13-induced constitutive NF-B activity not only allows the T1165 cells to establish peritoneal plasmacytomas without pristane preconditioning but also promote the development of disseminated disease with visceral involvement.

DISCUSSION
A key role of IL6 in myeloma pathogenesis is supported by the observations that STAT3 is constitutively active in primary myeloma cells and that inhibition of the IL6R/STAT3 pathway induces apoptosis in certain human myeloma cell lines in vitro (39). Furthermore, although intraperitoneal injections of pristane can induce plasmacytomas in the wild-type BALB/c mice (4), it fails to do so in IL6-null mice (40). Similarly, established IL6-dependent plasmacytoma cell lines, such as T1165 and B9, fail to form plasmacytomas when injected into IL6deficient mice (38). Collectively, these studies highlighted the potential significance of the IL6 pathway in myeloma pathogenesis and made it a prime target for therapeutic intervention.  However, the results of early clinical trials with IL6-blocking antibodies were disappointing (41) and have led to the suggestion that there are IL6-independent signaling pathways that play equally important role in the survival and proliferation of myeloma cells (2).
Genetic and epigenetic abnormalities in several genes involved in the NF-B pathway, including TRAF3, NIK, TRAF2, CYLD, BIRC2/BIRC3, CD40, NFKB1, NFKB2, LTBR, and TAC1, are seen in ϳ20% of patients with multiple myeloma and are associated with constitutive activation of the NF-B pathway (16,17). Inactivation of TRAF3 (TNF receptor-associated factor 3) and overexpression of NF-B-inducing kinase (NIK) are the two most common abnormalities associated with constitutive NF-B activation in myeloma samples and cell lines and have been shown to promote their survival (16,17). However, because TRAF3 and NIK also affect the MAPK signaling pathway (42)(43)(44), it was not clear whether deregulation of the NF-B pathways is solely responsible for the myeloma-promoting effects of the mutations observed in the above studies. In this study, we have used the viral proteins K13 and Tax as molecular tools to demonstrate that constitutive activation of the NF-B pathway is sufficient to confer IL6 independence on IL6-dependent plasmacytoma cells both in vitro and in vivo. Our conclusion is supported by the following lines of evidence. First, we demonstrate that the protective effect of K13 against IL6 withdrawal-induced apoptosis is associated with NF-B activation. Second, we did not observe any protection against IL6 withdrawal-induced apoptosis upon ectopic expression of an NF-B-defective mutant of K13. Third, ectopic expression of wild-type and mutant Tax constructs conferred protection against IL6 withdrawal-induced apoptosis, which correlated with their ability to activate the NF-B pathway. Fourth, the protective effect of K13 against IL6 withdrawal-induced apoptosis was reversed by treatment with NF-B inhibitors. Finally, ectopic expression of E8, a vFLIP that lacks the ability to activate NF-B, failed to confer protection against IL6 withdrawal-induced apoptosis. The latter finding also argued against the possibility that the protection conferred by K13 was due to its ability to act as a vFLIP (i.e. because of inhibition of FLICE/caspase 8).
Our study also suggests a possible mechanism by which K13induced NF-B protects plasmacytoma cells against IL6 withdrawal-induced apoptosis. The NF-B pathway has been shown to up-regulate the expression of a number of antiapoptotic members of the Bcl2 family (45). In this study, we observed that although the expression of Bcl-2, Bcl-xL, and Mcl-1 declined upon IL6 withdrawal in T1165-vector cells, it was maintained in T1165-K13 cells (Fig. 5F). Furthermore, ectopic expression of Bcl-2, Bcl-xL, and Mcl-1 protected T1165 cells against IL6 withdrawal-induced apoptosis (Fig. 5, G and H). Collectively, the above results suggest that IL6 signaling protects murine plasmacytomas cells against apoptosis by maintaining the expression of antiapoptotic members of the Bcl2 family. In the absence of IL6, the expression of antiapoptotic Bcl2 family members falls below a critical threshold, resulting in induction of apoptosis. In the K13-expressing plasmacytoma cells, however, NF-B signaling provides an alternative pathway for maintaining the expression of antiapoptotic Bcl2 family members, thereby protecting them from IL6 withdrawal-induced apoptosis. However, in addition to antiapoptotic Bcl2 family members, the K13-induced NF-B pathway is known to induce the expression of a number of other antiapoptotic and growth-promoting genes, such as BIRC3, IL8, CCL5, and GM-CSF (46). Thus, it is conceivable that additional genes induced by the NF-B pathway might contribute to the protective effect of K13 against IL6 withdrawal-induced apoptosis.
IL6 is one of the known NF-B target genes, and K13 is known to induce IL6 expression via NF-B activation (27,36,46). Therefore, we had expected that constitutive NF-B activation by K13 would confer protection against IL6 withdrawal by stimulating the production of endogenous IL6. A surprising finding of this study was that K13 protected against IL6 withdrawal-induced apoptosis without stimulating endogenous IL6 production. This conclusion was supported by our inability to detect murine IL6 in the supernatant of T1165-K13 cells either by ELISA or by a biological assay using fresh T1165 cells. Finally, the lack of phosphorylation of the downstream components of the IL6 signaling pathway, such as STAT1 and STAT3, and their resistance to JAK1/2 inhibitor INCB018424 ruled out the existence of intracellular IL6 signaling in the T1165-K13 cells.
The exact reason for the inability of K13-induced NF-B activation to up-regulate IL6 in the plasmacytoma cells is not clear at present. The IL6 promoter contains binding sites for several transcriptional factors. A recent study demonstrated that four transcriptional sites, NF-B, AP1, cAMP response element-binding protein, and CCAAT-enhancer-binding proteins, were collectively responsible for maximal IL6 expression in the IM9 myeloma cell line (47). Among these sites, the AP1binding site was shown to be the most important cis-regulatory site for constitutive IL6 expression (47). Importantly, this study also demonstrated that mutation of the NF-B-binding site had little effect on IL6 production in the IM9 cell line (47). Instead, NF-B required cooperative interaction with c-Jun, which constitutively occupies the AP1 site, for IL6 production (47). Interestingly, we have demonstrated recently that K13 selectively activates the NF-B pathway without concomitant JNK/AP1 activation (30). Thus, the lack of JNK/AP1 activation by K13 may provide a possible explanation for its inability to induce IL6 expression in the plasmacytoma cells. However, treatment with TNF␣, a known activator of the JNK/AP1 pathway, also failed to induce IL6 production in T1165 cells, suggesting the existence of additional molecular defects.
We observed that constitutive NF-B activation protected a majority of cells against IL6 withdrawal-induced apoptosis and led to rapid emergence of cells that could proliferate long-term in the absence of IL6. More importantly, intraperitoneal injection of these IL6-independent clones resulted in the development of abdominal plasmacytomas with visceral involvement without preconditioning by pristane. Collectively, these results suggest that NF-B activating mutations, such as those involving the TRAF3, NIK, TRAF2, CYLD, BIRC2/BIRC3, CD40, NFKB1, NFKB2, LTBR, and TAC1 genes, may not only contribute to the progression of myeloma to an IL6-independent phase but also to the development of disseminated disease with visceral involvement. The rapid emergence of IL6-independent clones that were dependent on NF-B signaling also attests to the remarkable plasticity and redundancy of the cellular survival pathways.
Our results also have significance for the development of targeted agents for the treatment of plasma cell disorders. Although early clinical trials with IL6-blocking antibodies were disappointing (41), there is a resurgence of interest in targeting IL6 signaling in myeloma. Several recent studies have described small molecule inhibitors of JAK1/2 with promising in vitro and in vivo activities against myeloma cell lines (48,49). However, our results show that NF-B activation confers IL6 independence via a pathway independent of JAK/STAT signaling and suggest that activation of the NF-B pathway may result in resistance to this class of drugs in plasma cell neoplasms, a notion supported by our results with INCB018424. Interestingly, we also observed that cells that had become IL6-independent because of activation of NF-B were extremely sensitive to NF-B inhibitors. Thus, combining a JAK1/2 inhibitor with an NF-B inhibitor may represent an attractive regimen that deserves further study for the treatment of plasma cell disorders. Another important finding of our study was that NF-B activation not only protected plasmacytoma cells against IL6 withdrawal-induced apoptosis but also made them resistant to dexamethasone, an agent commonly used for the treatment of plasma cell neoplasms. This association between emergence of IL6 independence and steroid resistance has important implications for the treatment of plasma cell disorders.
The significance of our results, however, is not limited to plasma cell disorders. HHV8-infected primary effusion lymphoma (PEL) cells display a plasmacytoid morphology and resemble myeloma cells in their gene expression profile and responsiveness to IL6, which is known to promote their growth in vitro and in vivo (50,51). Similar to plasma cell disorders, agents targeting the IL6 signaling pathway have been proposed for the treatment of PEL (52). However, as the NF-B pathway is constitutively active in PEL cells (23,53), it could potentially provide an IL6-independent pathway for the survival of PEL cells. Combination of agents targeting IL6 signaling with those targeting the NF-B pathway may represent a more promising approach for the treatment of these disorders.