Purpose: Activated B-cell diffuse large B-cell lymphoma (ABC-DLBCL) is an aggressive, poorly chemoresponsive lymphoid malignancy characterized by constitutive canonical NF-κB activity that promotes lymphomagenesis and chemotherapy resistance via overexpression of antiapoptotic NF-κB target genes. Inhibition of the canonical NF-κB pathway may therefore have therapeutic relevance in ABC-DLBCL. Here, we set out to determine whether dogs with spontaneous DLBCL have comparative aberrant constitutive NF-κB activity and to determine the therapeutic relevance of NF-κB inhibition in dogs with relapsed, resistant DLBCL.
Experimental Design: Canonical NF-κB activity was evaluated by electrophoretic mobility shift assays and immunoblot analyses, and NF-κB target gene expression was measured by quantitative real time PCR. Primary malignant canine B lymphocytes were treated with the selective IKK complex inhibitor NF-κB essential modulator-binding domain (NBD) peptide and evaluated for NF-κB activity and apoptosis. NBD peptide was administered intranodally to dogs with relapsed B-cell lymphoma and NF-κB target gene expression and tumor burden were evaluated pre- and post-treatment.
Results: Constitutive canonical NF-κB activity and increased NF-κB target gene expression were detected in primary DLBCL tissue. NBD peptide inhibited this activity and induced apoptosis of primary malignant B cells in vitro. Intratumoral injections of NBD peptide to dogs with relapsed DLBCL inhibited NF-κB target gene expression and reduced tumor burden.
Conclusions: This work shows that dogs with spontaneous DLBCL represent a clinically relevant, spontaneous, large animal model for human ABC-DLBCL and shows the therapeutic relevance of NF-κB inhibition in the treatment of ABC-DLBCL. These results have important translational relevance for ABC-DLBCL treatment in human patients. Clin Cancer Res; 17(14); 4661–71. ©2011 AACR.
Activated B-cell diffuse large B-cell lymphoma (ABC-DLBCL) is an aggressive, poorly chemoresponsive lymphoid malignancy characterized by constitutive canonical NF-κB activity that promotes lymphomagenesis and chemotherapy resistance via overexpression of antiapoptotic NF-κB target genes. We have investigated NF-κB as a therapeutic target by using the dog as a clinically relevant, spontaneous, animal model of DLBCL. We show that as in human ABC-DLBCL, malignant lymphocytes from dogs with DLBCL have constitutive canonical NF-κB activity and overexpress antiapoptotic genes. We show that the selective IKK inhibitor, NBD peptide inhibits NF-κB activity in vitro and leads to apoptosis of malignant B cells. We show in vivo that intratumoral injections of NBD peptide inhibit NF-κB gene expression and reduce tumor burden in dogs with DLBCL. These studies show the therapeutic relevance of NF-κB inhibition in vivo in a canine DLBCL model and have important translational relevance for the treatment of human ABC-DLBCL.
Diffuse large B-cell lymphoma (DLBCL) is the most common adult lymphoid malignancy with approximately 30,000 new cases diagnosed each year in the United States (1, 2). Gene expression profiling has revealed the presence of at least 3 subtypes of DLBCL, activated B-cell (ABC-DLBCL), germinal center B-cell (GCB-DLBCL), and primary mediastinal B-cell lymphoma (PMBL), that have distinct molecular signatures and different clinical outcomes (1, 3). ABC-DLBCL is the most aggressive subtype and is less responsive to conventional multiagent chemotherapy than GCB-DLBCL and PMBL. Even with the addition of rituximab, the overall survival for patients with ABC-DLBCL is 47% (4).
ABC-DLBCL is characterized by the presence of constitutive canonical NF-κB activity that drives expression of NF-κB target genes which promote lymphocyte proliferation (e.g., Cyclin D1 and D2) and inhibit apoptosis (e.g., Bcl-2, Bcl-xL,A1, c-FLIP, and XIAP; refs. 5, 6). Aberrant canonical NF-κB activity contributes to lymphomagenesis and chemoresistance by promoting malignant cell survival and proliferation (7, 8). In health, NF-κB activation is highly regulated. NF-κB dimers are held inactive in the cytoplasm through their association with inhibitory IκB family members (9). Engagement of cell surface receptors by many different infectious and inflammatory stimuli leads to phosphorylation and activation of the IKK complex, which phosphorylates IκBα and targets it for ubiquitination and proteosomal degradation. This allows NF-κB dimers to translocate to the nucleus where they bind to target gene promoters and enhancers and initiate transcription (8). Somatic activating mutations in genes encoding regulatory proteins upstream of the IKK complex have been identified in patients with ABC-DLBCL and lead to constitutive phosphorylation and activation of the IKK complex (5, 10, 11). Constitutive, canonical NF-κB activity is essential for ABC-DLBCL cell survival (3, 7) and inhibition of this pathway with small molecule inhibitors of IKK, IκBα super repressors, proteosome inhibitors, or inhibitors upstream of the IKK complex promotes cell-cycle arrest, chemotherapeutic sensitivity, and apoptosis in ABC-DLBCL cell lines (3, 7, 12). Taken together, these findings suggest that targeted inhibition of aberrant NF-κB activity represents a promising therapeutic strategy for patients with primary or relapsed ABC-DLBCL.
The high-quality draft genome sequence of the dog has revealed its close phylogenetic relationship with man, emphasizing the potential benefit of canine models in identifying disease genes and evaluating response to novel therapies (13–16). In particular, the dog is being increasingly recognized as a clinically relevant, spontaneous large animal model for human non-Hodgkin's lymphoma (NHL; refs. 17–19). NHL is the most common, spontaneous, hematopoietic malignancy in dogs, with an annual incidence of 30/100,000 (20). DLBCL is the most common subtype of canine NHL and shares similar biologic, behavioral, molecular, and genetic characteristics with DLBCL in humans (17, 20–22). Dogs with DLBCL are treated with the same cytotoxic agents used in human DLBCL patients that inhibit cell division and induce apoptosis. However, as in human patients, clinical remission is not maintained and 85% to 90% of canine patients relapse with lethal, drug-resistant lymphoma within 6 to 9 months of initial diagnosis and treatment (19, 23).
NF-κB essential modulator (NEMO)-binding domain (NBD) peptide is a selective inhibitor of the IKK complex that consists of 11 amino acids located at the carboxy terminus of the catalytic IKKβ subunit that binds to the scaffold protein NEMO (24). The core of 6 amino acids in the NBD is also present in IKKα and facilitates its association with NEMO (24–26). NBD peptide inhibits the interaction of both IKKα and IKKβ with NEMO and prevents assembly of the IKK complex (25). Fusion of NBD peptide to protein transduction domains, such as that within the Drosophila antennapedia protein enables it to enter cells and effectively inhibit canonical NF-κB activation in response to TNFα, lipopolysaccharide and toll-like receptor ligation (24, 25, 27). Systemic administration of NBD peptide results in potent antiinflammatory effects in animal models of inflammation (24, 28–32). NBD peptide also inhibits constitutive NF-κB activity in tumor cell lines, including pancreatic carcinoma, squamous cell carcinoma, melanoma, cutaneous T-cell lymphoma, and mantle cell lymphoma, and sensitizes these cells to TNF-related apoptosis-inducing ligand or TNFα-induced apoptosis (33–37). Furthermore, NBD peptide inhibits NF-κB activation that occurs in response to DNA damage induced by cytotoxic chemotherapeutics (38). Taken together, these findings suggest that targeted inhibition of canonical NF-κB activity by using NBD peptide may represent an effective therapeutic strategy in patients with ABC-DLBCL.
Before evaluating the therapeutic effects of NBD peptide in human patients with ABC-DLBCL, we conducted studies to evaluate its ability to inhibit constitutive NF-κB activity in dogs with spontaneous B-cell lymphoma. We show that constitutive canonical NF-κB activity occurs in the malignant lymph nodes of dogs with DLBCL and that this is associated with overexpression of antiapoptotic NF-κB target genes. We show that NBD peptide inhibits constitutive NF-κB activity and induces apoptosis of canine malignant B cells in vitro. Furthermore, we show that administration of NBD peptide to dogs with relapsed B-cell lymphoma inhibits the expression of NF-κB target genes and leads to a reduction in tumor burden. These findings provide proof of concept that NBD peptide can inhibit constitutive NF-κB activity in a clinically relevant spontaneous canine model of DLBCL and begin to pave the way for phased clinical trials in humans with ABC-DLBCL.
Materials and Methods
Cells and reagents
Canine lymphocytes were cultured in complete RPMI media containing 10% FBS. The human GCB-DLBCL cell line SUDHL-6 was maintained in complete RPMI media, and the human ABC-DLBCL cell line OCI-Ly10 was maintained in complete Iscove's modified essential medium containing 20% FBS. Cell lines were obtained from Dr. Anne Novak (Mayo Clinic Cancer Center, Rochester, MN). Malignant lymph node samples were collected from dogs with either primary or relapsed DLBCL following approval from the University of Pennsylvania's Institutional Animal Care and Use Committee. The histopathologic diagnosis of DLBCL and the cytologic diagnosis of large B-cell lymphoma were made by board-certified veterinary pathologists (M.G. and A.D., see Acknowledgements) and by a board-certified clinical pathologist (R.P.), respectively. NBD peptide was synthesized and purified by Dr. James I. Elliott (at the Howard Hughes Medical Institute Biopolymer-Keck Foundation Biotechnology Resource Laboratory, Yale University, New Haven, CT) using standard tertbutoxycarbonyl chemistry, cleavage with hydrofluoric acid, and purification by reversed-phase high-performance liquid chromatography (26). TNFα was purchased from Sigma-Aldrich.
Western blot analyses
Cells were lysed in 50 mmol/L Tris-HCL containing 1% NP-40, 150 mmol/L NaCl, 2.5 mmol/L EDTA, 5% glycerol, a 1:50 dilution of protease inhibitor cocktail, and 1:50 dilutions of phosphatase inhibitor cocktails 2 and 3 (Sigma-Aldrich). Protein concentrations were determined by micro BCA assay (Thermo Scientific). Proteins were separated by SDS-PAGE (10%) and transferred to polyvinylidene difluoride membrane. Membranes were probed with polyclonal rabbit anti-human antibodies against phospho-IKKα/β (16A6), pan IKKβ (L570), phospho-IκBα (14D4), and β-actin (4,967; Cell Signaling) or rabbit anti-human pan IκBα (C-21; SantaCruz Biotechnology). A horseradish peroxidase-conjugated donkey anti-rabbit IgG was used as the secondary detection antibody (Amersham). Blots were developed by using ECL Plus (Amersham) or SuperSignal West Femto (Thermo Scientific).
Electrophoretic mobility shift assay
Whole cell extracts of malignant canine lymphocytes were prepared as previously described (26). Electrophoretic mobility shift assays (EMSA) were done by using 5 μg protein extract and a palindromic NF-κB binding sequence probe (Santa Cruz Biotechnology) as previously described (26). EMSA supershift assays were done by using 10 μg of protein extract and polyclonal rabbit anti-human p65 (sc-109X), p50 (sc-114X), and c-Rel (sc-70X) antibodies or control rabbit IgG (sc-2027; Santa Cruz Biotechnology).
Quantitative reverse transcription PCR
Total RNA extraction was done by using the RNeasy Mini Kit (Qiagen). Reverse transcription was done by using random hexamers and Superscript II reverse transcriptase (Invitrogen Corp.). Transcript sequences for canine A1, c-FLIP, Cyclin D1, and IκBα were obtained from the National Center for Biotechnology Information (http://www.ncbi.nih.gov/Genbank) and were analyzed for secondary DNA structure by using M-Fold (http://frontend.bioinfo.rpi.edu/applications/mfold/cgi-bin/dna-form1.cgi). Primers were designed by using Primer 3 software (http://frodo.wi.mit.edu/primer3/) with the maximum self-complementarity score set at 5 and the maximum 3 self-complementarity score set to 0 to minimize primer-dimer formation (Table 1). Primer sequences for canine Bcl-2, XIAP, and β-actin have been previously described (39). Quantitative real time PCR (qRT-PCR) was done by using SYBR Green (Fermentas). Samples were run in triplicate by using standard conditions on an ABI 7500 sequence detector (Applied Biosystems) and data were analyzed by using β-actin as an endogenous control. Dissociation curves were done after each experiment to confirm the specificity of product amplification.
Immunohistochemical analysis was done on formalin fixed, paraffin-embedded tissue sections of lymph nodes from healthy dogs and dogs with DLBCL. Sections were deparaffinized in xylene, rehydrated, and boiled in sodium citrate buffer to unmask antigens. Endogenous peroxidase was blocked with 3% hydrogen peroxide. Immunohistochemistry was done by using a rabbit anti-human p65 antibody (C22B4; Cell Signaling) or rabbit isotype control (sc-2027; Santa Cruz Biotechnology) and the Vectastain Elite ABC Kit (Vector Laboratories). Sections were developed by using 3,3′-diaminobinzidine and counterstained with hematoxylin. Slides were viewed by using a Nikon E600 infinity corrected upright microscope. Bright-field images were acquired by using a Nikon Digital Sight DS-Fi1 color camera by using NIS-Element BR3.0 for image analysis.
In vitro NBD peptide treatment of malignant B lymphocytes
Freshly isolated or cryopreserved malignant lymphocytes from lymph nodes of dogs with DLBCL were seeded at 4 × 106 cells/mL and treated with 100 μm of either NBD or mutant peptide in dimethyl sulfoxide (DMSO). After 12 hours, whole cell extracts were evaluated by immunoblot for canonical NF-κB activity as described above and for apoptosis by flow cytometry. The human ABC- and GCB-DLBCL cell lines OCI-Ly10 and SUDHL-6 were seeded at 0.5 × 106 cells/mL and treated with 0, 25, or 50 μmol/L NBD peptide. After 24 hours, cell death was evaluated by Trypan Blue.
Detection of apoptotic cell death was done by using the PE Annexin-V Apoptosis Detection Kit (BD Biosciences). Cells were acquired on a FACSCalibur cytometer (BD Biosciences) and analyzed by using TreeStar FlowJo software. Immunophenotyping of primary lymphoma cells was done by using the following antibodies: Fluorescein isothiocyanate (FITC)-conjugated rat anti-canine CD3 (Serotec), adenomatous polyposis coli (APC)-conjugated mouse anti-human CD79a (BD Pharmingen) and mouse anti-canine CD21-like molecule (Serotec). A FITC-labeled goat anti-mouse IgG secondary antibody was used to detect anti-CD21 antibody. For CD79a staining, cells were fixed with 1% paraformaldehyde and then permeabilized with 0.1% saponin before staining.
All in vivo studies were done following the approval from the University of Pennsylvania's Institutional Animal Care and Use Committee and the University of Pennsylvania School of Veterinary Medicine Clinical Review Board. Client-owned dogs were enrolled with the following inclusion criteria: (i) cytologic confirmation of relapsed large B-cell lymphoma, (ii) stage III–V, substage a, with at least 1 set of bilateral, enlarged, measurable, malignant lymph nodes, (iii) life expectancy of greater than 1 month, (iv) no concurrent systemic disease, (v) adequate hematologic, renal, and hepatic function, and (vi) the presence of constitutive NF-κB activity within malignant lymph nodes as determined by EMSA. All dogs enrolled in the pilot trial had been previously treated with a range of rescue chemotherapeutic agents including L-asparaginase, cyclophosphamide, vincristine, prednisone, lomustine, doxorubicin, mechlorethamine, procarbazine, dacarbazine, and total body irradiation. Signed-informed consent was obtained from all owners before entry into the study. Baseline evaluation included a complete medical history, full clinical examination, complete blood count (CBC), chemistry screen (CS) and urinalysis, malignant lymph node cytology, and flow cytometric immunophenotyping by using a basic panel of antibodies including CD3, CD21, and CD79a. Eligible dogs received either 5 or 10 mg NBD peptide/100 g malignant lymph node mass. Node mass was calculated based on 2 dimension caliper measurements which were averaged and considered the diameter of the lymph node sphere. Volume was determined based on V = 2/3πr3. On the basis of our previous assessments, 1 cm3 of malignant lymph node mass is equivalent to ∼379 mg. Peptide was injected directly into 1 malignant node and the same node was biopsied 24 hours later. qRT-PCR for NF-κB target gene expression was done on node tissue taken 24 hours post-NBD peptide treatment and was normalized to values obtained pre-NBD peptide. No control dogs were included in this pilot study. Following the post-treatment biopsy, patients received rescue chemotherapy as determined by their primary attending oncologist. All dogs received a follow-up full clinical examination plus CBC, CS, and urinalysis 1-week post-NBD peptide injection. Mass of the NBD-treated lymph node and the contralateral, malignant, untreated lymph node was calculated before, and 1-week post-NBD peptide administration.
Constitutive canonical NF-κB activity occurs in dogs with DLBCL
To determine whether constitutive NF-κB activity is present in dogs with spontaneous, histologically confirmed DLBCL, EMSA was done on biopsy samples taken from the peripheral lymph nodes of 13 dogs with DLBCL at the time of diagnosis and prior to chemotherapy. Constitutive NF-κB activity was detected in all 13 samples. In contrast, NF-κB activity was not detected in normal lymph nodes from healthy dogs (Fig. 1A). To determine whether constitutive NF-κB activity was associated with the canonical pathway, malignant lymph node tissue from 11 dogs with newly diagnosed, histologically confirmed DLBCL was analyzed by EMSA supershift by using antibodies directed against p50, p65, and c-Rel. In 7 dogs, p50, p65, and c-Rel were all present in the active NF-κB complexes (Fig. 1B). In 2 dogs, only p50 and p65 were present, and in 2 dogs, only p50 was identified (data not shown). To further confirm the presence of constitutive canonical NF-κB activity, formalin-fixed, paraffin-embedded lymph node tissues from 6 dogs with DLBCL were evaluated for the presence of nuclear p65 by immunohistochemistry (Fig. 1C). All tissue sections had abundant cytoplasmic and nuclear-localized p65 whereas 4 healthy canine lymph node sections had minimal p65 staining.
To determine whether the detected canonical NF-κB activity was associated with constitutive IKK activity, the phosphorylation status of the IKK complex and the inhibitory IκBα protein were evaluated by immunoblot (Fig. 1D). p-IKKβ and p-IκBα were detected in 8 of 9 lymph node biopsy samples taken from dogs with histologically confirmed, untreated DLBCL with constitutively active NF-κB as determined by EMSA. In contrast, p-IKKβ and p-IκBα were only detected at very low levels in normal lymph nodes taken from healthy dogs (Fig. 1D). Taken together, these findings show that constitutive canonical NF-κB activity occurs in the malignant tissue of dogs with DLBCL, and that in the majority of dogs this activity is associated with IKK activation and downstream IκBα phosphorylation. These data parallel the findings in human ABC-DLBCL and suggest that dogs with DLBCL represent a clinically relevant, spontaneous large animal model in which to evaluate the therapeutic effects of NF-κB inhibition in the treatment of ABC-DLBCL.
Antiapoptotic NF-κB target genes are upregulated in canine DLBCL
To determine whether NF-κB target genes are upregulated in canine DLBCL, malignant lymph node tissue from 14 dogs with histologically confirmed DLBCL, and constitutive NF-κB activity was evaluated by qRT-PCR for Bcl-2, c-FLIP, and XIAP gene expression (Fig. 2). Tissue samples were obtained at the time of diagnosis and prior to chemotherapy. Using healthy canine lymph node tissue as a normalizing control, statistically significant increases in Bcl-2 gene expression were identified in 7 dogs. In addition, c-FLIP and XIAP gene expression was significantly increased in 6 and 7 dogs, respectively. These results suggest that constitutive canonical NF-κB activity in canine DLBCL promotes the overexpression of antiapoptotic NF-κB target genes. This finding parallels that in human patients with ABC-DLBCL and indicates that constitutive canonical NF-κB activity in the dog may contribute to lymphomagenesis and chemoresistance.
NBD peptide inhibits constitutive NF-κB activity and promotes apoptosis in DLBCL cells in vitro
To determine whether NBD peptide can inhibit constitutive phosphorylation of IKK and prevent downstream IκBα phosphorylation, cells from malignant lymph nodes of dogs with DLBCL were treated in vitro with either 100 μmol/L NBD peptide or 100 μmol/L mutant peptide for 12 hours and then analyzed for the presence of p-IKKβ and p-IκBα by immunoblot (Fig. 3A and B). Cells treated with NBD peptide showed a marked reduction of p-IKKβ compared with untreated cells, and those treated with either mutant peptide or DMSO vehicle control. NBD peptide treatment also resulted in complete inhibition of IκBα phosphorylation (Fig. 3B). In contrast, treatment with mutant peptide or DMSO (vehicle control) did not inhibit IκBα phosphorylation.
To determine the effects of NF-κB inhibition on DLBCL cell apoptosis, malignant lymphocytes harvested from canine DLBCL tumors were treated in vitro with NBD or mutant peptide and analyzed by flow cytometry (Fig. 3C). After 12 hours incubation with NBD peptide, more than 50% of DLBCL cells stained positive for Annexin-V while only 15% of cells treated with mutant peptide appeared apoptotic. Comparable results were identified in malignant cells taken from the lymph nodes of 3 dogs with DLBCL. In contrast, when peripheral blood mononuclear cells (PBMC) isolated from healthy dogs were treated with either 100 μmol/L NBD or mutant peptide for 12 hours, less than 5% of PBMCs stained positive for Annexin-V (Fig. 3D). Collectively, these data show that NBD peptide inhibits constitutive NF-κB activity in canine DLBCL cells, leading to selective, rapid apoptosis of malignant cells.
To confirm a selective cytotoxic effect of NBD peptide on human ABC-DLBCL cells, OCI-Ly10 cells and the GCB-DLBCL cell line SUDHL-6 were treated with NBD peptide for 24 hours and the percentage increase in cell death compared with untreated cells was calculated. At 24 hours, NBD peptide induced a dose-dependent increase in cell death of the ABC-DLBCL cell line, OCI-Ly10. In contrast, NBD peptide did not induce cell death in the GCB-DLBCL cell line at either concentration used (Fig. 3E).
In vivo administration of NBD peptide inhibits NF-κB target gene expression and leads to a reduction in tumor burden in dogs with relapsed, chemoresistant large B-cell lymphoma
To determine whether NBD peptide can inhibit NF-κB target gene expression in malignant lymphoid tissue in vivo, privately owned dogs with relapsed, chemoresistant large B-cell lymphoma were enrolled to a small pilot study (Fig. 4). Eligibility criteria included the presence of histologically or cytologically confirmed large B-cell lymphoma and constitutive NF-κB activity in malignant lymph nodes as determined by EMSA. Response to NBD peptide was determined by evaluating NF-κB target gene expression pre- and 24 hours post-NBD peptide administration. Clinical response to NBD peptide was determined by comparing the mass of the injected lymph node before NBD peptide and 1 week after administration of peptide plus rescue chemotherapy. The mass of the contralateral malignant lymph node that was not injected with NBD peptide but was exposed to systemic rescue chemotherapy was also determined at both time points and the change in lymph node mass was calculated for both nodes and compared. Six eligible dogs were enrolled to the study, 3 dogs received 5 mg NBD peptide/100 g of node tissue and 3 received 10 mg NBD peptide/100 g node tissue via a single intranodal injection. At the time of enrollment, qRT-PCR analysis of lymph node tissue revealed that all dogs overexpressed at least 2 of 6 NF-κB target genes that promote cellular proliferation and inhibit apoptosis when compared with normal lymph node tissue (Fig. 5A left). 24 hours post-NBD peptide administration 1 of 3 dogs receiving 5 mg NBD peptide/100 g nodal tissue showed a reduction in the expression of 4/6 NF-κB target genes within the malignant node when compared with pre-NBD treatment gene expression values. At the higher dose of NBD peptide, all 3 dogs showed a reduction in the expression of at least 3/6 NF-κB target genes including Bcl-2, Cyclin D1, and IκBα when compared with pre-NBD treatment values (Fig. 5A right).
Response to NBD peptide was determined by comparing the change in mass of the NBD peptide–treated lymph node with change in mass of the contralateral, malignant, noninjected lymph node. Results were available for 4 of 6 treated dogs (Fig. 5B). Rescue chemotherapy protocols administered to each dog after NBD peptide treatment varied among dogs and were determined by the referring oncologist. In 3 of 4 dogs, the NBD peptide–treated lymph node showed between a 1.5- and 6-fold reduction in mass when compared with the contralateral, malignant lymph node that did not receive NBD peptide. Two dogs (dogs 7 and 8) were treated on a compassionate basis and given intranodal injections of 10 mg NBD/100 g tumor into 8 peripheral enlarged lymph nodes, resulting in cumulative NBD doses of 0.33 and 0.8 mg/kg body weight, respectively. This amendment to the original protocol was approved by the University of Pennsylvania's Institutional Animal Care and Use Committee. At enrollment, each dog overexpressed at least 3 NF-κB target genes (Fig. 5A left). 24 hours after NBD peptide administration, reductions in NF-κB target gene expression were observed in 3/6 and 6/6 evaluated genes, respectively (Fig. 5A right). In these 2 dogs, a reduction in lymph node mass was observed in 3/8 and 4/8 NBD-treated lymph nodes (Fig. 5C). Despite NBD peptide and systemic chemotherapy, the remaining lymph nodes continued to increase in size. In both dogs, the lymph nodes that showed a reduction in mass following NBD peptide treatment were the smallest nodes at the time of treatment.
Aberrant, constitutive, canonical NF-κB activity is recognized as a key contributor to oncogenesis and chemotherapy resistance in many different cancers and as such represents a logical target for inhibition in cancer therapy. Although nearly 800 inhibitors of NF-κB have been identified (40), their ability to inhibit constitutive NF-κB activity in spontaneous cancers in vivo and the therapeutic relevance of such inhibition in patients with cancer is unknown.
In this study, we first set out to determine whether dogs with DLBCL represent an appropriate, spontaneous, large animal model in which to evaluate the therapeutic effects of NF-κB inhibition. DLBCL in the dog is often characterized by total effacement of lymph node architecture with large malignant B cells that have a high-mitotic index and multiple, prominent nucleoli (41). This malignancy in dogs is frequently associated with overexpression of c-myc that occurs as a result of chromosomal translocations that place the oncogene under control of the IgH promoter (17). Although these histocytologic and cytogenetic changes are also found in ABC-DLBCL in humans, it is unknown whether canine DLBCL is characterized by constitutive canonical NF-κB activity. Here, we show that dogs with histologically confirmed DLBCL have constitutive canonical NF-κB signaling in malignant lymphoid tissue at the time of diagnosis. In most cases evaluated, p50/p65 and p50/c-Rel heterodimers were present in the activated complex. Only 2 biopsy samples had evidence of active p50 in the apparent absence of active p65 or c-Rel suggesting the presence of p50 homodimers or complexes with RelB in these patients. These differences may be attributed to the specific mechanism(s) responsible for constitutive NF-κB activity in each tumor sample, as the identity and relative abundance of active homo- and heterodimers depends on the nature of the activating signal or pathway aberrancy (42, 43). Furthermore, as in human ABC-DLBCL patients, constitutive activity was associated with phosphorylation of IKKβ and IκBα in all but 1 dog evaluated, indicating that in the majority of cases signals leading to constitutive activity occur upstream of the IKK complex (7, 10). These findings confirm on a molecular level that human ABC-DLBCL and canine DLBCL have similar aberrant NF-κB signaling and that the dog may serve as a clinically relevant large animal model in which to study the therapeutic effects of NF-κB pathway inhibition in ABC-DLBCL.
Although constitutive NF-κB activity was present in the malignant lymph nodes of all dogs with untreated DLBCL, the NF-κB target genes Bcl-2, c-FLIP, and XIAP were overexpressed in only half of the biopsy samples. These findings serve to underline the multiple layers of complexity involved in the regulation of NF-κB target gene expression. First, the particular combination of activated NF-κB family members influences the target gene expression profile, in part because these family members have different binding affinities for different gene promoters. Although NF-κB activation originates upstream of IKK in these dogs, the specific activation triggers have not been investigated and may differ between dogs, resulting in different NF-κB family member usage and different target gene expression signatures. However, in this small dataset, we did not find any correlation between the expression of NF-κB target genes and activation of specific NF-κB family members. Second, other signaling pathways that activate additional transcription factors such as AP-1 and ETS may be concurrently active in these tumors. These transcription factors together with cofactors that modify NF-κB activity influence gene activation and repression, leading to variable gene expression profiles (44, 45). Taken together these points highlight the complex and multifaceted nature of NF-κB regulated gene expression. A much larger sample set of histologically confirmed, DLBCL biopsies with complete gene expression profiling together with a thorough evaluation of activated NF-κB family members will be needed to identify specific NF-κB gene expression signatures.
In the second part of this study, we show that NBD peptide can inhibit constitutive IKKβ and IκBα phosphorylation in DLBCL biopsy samples which leads to increased apoptosis of malignant lymphocytes. Furthermore, in a small clinical pilot study, NBD peptide showed a dose-dependent inhibition of NF-κB target gene expression in dogs with relapsed, refractory large B-cell lymphoma. In 2 dogs receiving the lower NBD peptide dose (dogs 1 and 3), all NF-κB target genes increased up to 4-fold 24 hours post-NBD peptide suggesting that constitutive NF-κB activity in these dogs may be exerting a transcriptional repressive effect. No correlation was identified between 24-hour gene expression profile results and change in lymph node mass 7 days later.
In 3 of 4 dogs, intranodal administration of NBD peptide led to a marked reduction in tumor mass when compared with the contralateral, malignant lymph node that did not receive NBD peptide, although it is unclear whether NBD peptide alone, or acting synergistically with rescue chemotherapy exerted this effect. In dogs that received multinode injections of NBD peptide, the apparent effects on tumor mass were variable. Although most nodes continued to increase in size despite NBD treatment and rescue chemotherapy, several nodes in both dogs showed a marked reduction in tumor mass. In both cases, responding nodes were smaller than nonresponsive nodes at the time of treatment. These results raise the possibility that the NBD peptide dose and its ability to diffuse within malignant nodes may influence its ability to exert a measurable therapeutic effect.
Given the central role of the IKK complex in canonical NF-κB activation, IKK inhibitors are being investigated as novel therapeutic agents in both solid and hematologic malignancies that depend upon constitutive NF-κB activity for their survival. IKKβ inhibitors have been developed to specifically block canonical NF-κB signaling; however, in the presence of selective IKKβ inhibition, compensatory IKKα activity can drive NEMO-dependent canonical NF-κB activity in human DLBCL cells (46). This finding suggests that inhibitors of both IKKα and IKKβ activity (such as NBD peptide) will provide more complete inhibition and have greater therapeutic efficacy than IKKβ inhibitors alone. Furthermore, agents such as NBD peptide that specifically target the scaffolding molecule NEMO are less likely than kinase inhibitors to exhibit off-target effects on other intracellular signaling pathways (27).
Previous studies evaluating the safety and efficacy of NBD peptide as an antiinflammatory agent in vivo have shown that both short- and long-term administration of NBD peptide to piglets and rodents at doses ranging from 0.75 to 20 mg/kg is well tolerated (24, 28–31). In this small pilot study, NBD peptide did not cause any systemic toxicity and there were no significant changes in serum chemistries or hematologic profiles over the 10-day assessment period that could be attributable to NBD peptide administration. However, the dose of NBD peptide administered on a mg/kg body weight basis was very low. However, 2 dogs treated with the higher dose of NBD peptide developed submandibular abscesses within 1 week of NBD injection into prescapular lymph nodes. In both cases, the commensal skin bacteria Staphalococcus aureus was cultured and successfully treated with drainage and broad-spectrum antibiotics. In these cases, it is possible that commensal skin bacteria introduced with NBD peptide were able to colonize the necrotic node and that inhibition of TLR signaling by NBD peptide may have contributed to this process.
In this pilot study, NBD peptide was administered intranodally rather than systemically to first determine whether it could inhibit constitutive NF-κB activity in malignant lymphocytes in vivo. On the basis of the favorable in vitro and in vivo results, we are now actively recruiting dogs with ABC-DLBCL to a phase I dose escalation clinical trial in which NBD peptide will be administered systemically via the intravenous route. Although the amount of peptide necessary to achieve complete inhibition of NF-κB activity in a dog or human are likely to be high, it is unknown whether complete inhibition is required to see a therapeutic effect when used in combination with chemotherapy.
Taken together, the results reported here provide the first in vivo evidence that NBD peptide can inhibit antiapoptotic target gene expression driven by constitutive NF-κB activity and that this inhibition, either alone or in combination with cytotoxic therapy, can lead to improved clinical responses in dogs with ABC-like DLBCL. Given our reported findings that show the dog is a highly relevant, spontaneous clinical model for ABC-DLBCL in humans, the therapeutic effects of NF-κB inhibition seen in the canine cancer patient are likely to hold high translational relevance to human patients with ABC-DLBCL.
Disclosure of Potential Conflicts of Interest
M.J. May holds a patent for the NBD peptide and A.S. Baldwin is the founder of TheraLogics and therefore has commercial interests. All other authors declare no competing financial interests.
American Cancer Society ACS-IRG (N.J. Mason), American Kennel Club Canine Health Foundation (N.J. Mason), the Mari Lowe Center for Comparative Oncology (N.J. Mason), and the Canine Hereditary Cancer Consortium/NIH UC2 CA148149 (N.J. Mason). A. Gaurnier-Hausser is supported by the Ruth L. Kirschstein grant/NIH T32 CA 09140.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Received December 20, 2010.
- Revision received May 2, 2011.
- Accepted May 18, 2011.
- ©2011 American Association for Cancer Research.