B-cell activating factor and v-Myc myelocytomatosis viral oncogene homolog (c-Myc) influence progression of chronic lymphocytic leukemia

Abstract

Mice bearing a v-Myc myelocytomatosis viral oncogene homolog (c-Myc) transgene controlled by an Ig-alpha heavy-chain enhancer (iMyc(Cα) mice) rarely develop lymphomas but instead have increased rates of memory B-cell turnover and impaired antibody responses to antigen. We found that male progeny of iMyc(Cα) mice mated with mice transgenic (Tg) for CD257 (B-cell activating factor, BAFF) developed CD5(+) B-cell leukemia resembling human chronic lymphocytic leukemia (CLL), which also displays a male gender bias. Surprisingly, leukemic cells of Myc/Baff Tg mice expressed higher levels of c-Myc than did B cells of iMyc(Cα) mice. We found that CLL cells of many patients with progressive disease also expressed high amounts of c-MYC, particularly CLL cells whose survival depends on nurse-like cells (NLC), which express high-levels of BAFF. We find that BAFF could enhance CLL-cell expression of c-MYC via activation the canonical IκB kinase (IKK)/NF-κB pathway. Inhibition of the IKK/NF-κB pathway in mouse or human leukemia cells blocked the capacity of BAFF to induce c-MYC or promote leukemia-cell survival and significantly impaired disease progression in Myc/Baff Tg mice. This study reveals an important relationship between BAFF and c-MYC in CLL which may affect disease development and progression, and suggests that inhibitors of the canonical NF-κB pathway may be effective in treatment of patients with this disease.

Fig. 1.

Male Myc/Baff mice develop a monoclonal CD5⁺CD3⁻B220^low cell population resembling CLL. (A) Blood was collected from 8-mo-old WT, Baff-Tg, Myc-Tg, and Myc/Baff-Tg mice and was stained with CD5–allophycocyanin (APC) and B220-FITC antibodies and analyzed by flow cytometry. Shown is a representative dot plot from 1 of 12 mice per genotype. (B) Early clonal expansion of B cells in Myc/Baff-Tg mice was determined by Southern blot analysis of genomic DNA from splenocytes of mice age 8 mo or older using a J_H probe. Monoclonal bands are marked by an asterisk. GL, germline bands. (C) Survival (Kaplan–Meier) plots of cohorts of 12 age-matched male mice of the indicated genotypes.

Fig. 2.

Myc/Baff mice exhibit splenomegaly, increased splenic FDG uptake, and disrupted microarchitecture. (A) Representative FDG-PET scans of Myc and Myc/Baff mice. The arrows indicate spleen locations (regions of interest, ROI) (Left). Average FDG intensity of the ROI, was determined for two 1-y-old mice of each genotype (Right). (B) Average spleen weight of 8-mo-old mice of the indicated genotypes. Data are means ± SEM (n = 3). (C) Paraffin-embedded spleen sections from 8-mo-old male mice of the indicated genotypes were stained with H&E (n = 3, magnification: 200×). Arrows indicate the center of the white pulps that are disrupted in Myc/Baff mice.

Fig. 3.

BAFF elevates expression of survival and proliferation genes in leukemic cells. (A) B220⁺ B cells and CD5⁺CD3⁻B220^low leukemic cells were isolated from 8-mo-old Myc and Myc/Baff littermates, respectively. RNA was extracted and analyzed by qRT-PCR for expression of the indicated antiapoptotic genes. Results are means ± SEM (n = 3). *P < 0.05. (B and C) Splenic B220⁺ cells from WT, Baff, and Myc mice and CD5⁺CD3⁻B220^low leukemic cells from 8-mo-old Myc/Baff mice were divided into cytosolic (B) and nuclear (C) fractions that were gel separated and analyzed by immunoblotting. Results are representative of three independent experiments. (D) Blood mononuclear cells from 4-mo-old mice of the indicated genotypes were stained for CD5-APC, B220-phycoerythrin (PE), and Ki-67–FITC. The histogram shows the average percentages ± SEM of Ki-67⁺ cells (n = 5). (E) Paraffin-embedded spleen sections from 8-mo-old mice of the indicated genotypes were subjected to TUNEL staining. Numbers of apoptotic cells per field were determined from five age-matched mice per group. Results are means ± SEM. (F) Primary mouse T and B cells were isolated from WT spleens, and leukemic cells were isolated from Myc/Baff mice. Cell viability was determined by PI and DIOC6 staining of triplicate samples. Results are means ± SEM (n = 3).

Fig. 4.

BAFF enhances c-MYC mRNA expression through the IKK/N-κB pathway. (A) c-MYC mRNA expression was examined by microarray (Left) and qRT-PCR (Right) analyses of B220⁺ cells and CD5⁺CD3⁻B220^low cells isolated from three 8-mo-old Myc or Myc/Baff male mice, respectively. Results are means ± SEM (n = 3). (B) Primary B cells of the indicated genotypes and leukemic cells from Myc/Baff mice were isolated by magnetic separation and analyzed for c-MYC expression by immunoblotting. (C) Primary CLL cells were pretreated with BMS345541 (1 μM) for 20 min and then were incubated with recombinant human (rh) BAFF (200 ng/mL). Total RNA was isolated 6 h later, and c-MYC mRNA was measured by qRT-PCR. Results are averages ± SEM of three independent experiments with P values indicated. (D) Primary CLL cells were cultured and treated as in C. Cell lysates were collected 12 h later and analyzed for c-MYC expression by immunoblotting. (E) Primary CLL cells were infected with lentivirus-encoding GFP and shRNA specific for IKKβ mRNA or a scrambled control. GFP⁺ cells were isolated by flow cytometry and treated with BAFF. Protein lysates were analyzed by immunoblotting, quantified with Image J, and normalized to GFP expression. (F) Primary CLL cells were treated with BAFF for 2 h before ChIP analysis was performed on the c-MYC promoter. Specific primers flanking the predicted NF-κB consensus-binding sites were used for real-time PCR.

Fig. 5.

Elevated c-MYC expression is associated with CLL progression and NLC dependency. (A) Relative c-MYC mRNA expression in leukemia cells from a cohort of 166 CLL patients. The y axis represents the normalized log₂-transformed mRNA levels from Affymetrix GeneChips. (B) Elevated c-MYC expression is associated with more rapid disease progression. Patients were classified into three groups (low: 6–8; medium: 8–9.4; high: above 9.4) by a recursive partitioning method based on log₂-transformed c-MYC mRNA from the Affymetrix array, and their progression from CLL diagnosis to the start of treatment was plotted as a function of time. (C) Primary CLL cells (five each with high or low c-MYC expression) were cultured in vitro, and their viability was determined by DIOC6 and PI staining. Results are means ± SEM (n = 5 cells per group). (D) High c-MYC expression is associated with NLC dependency. c-MYC protein levels in 39 primary CLL specimens were examined by immunoblotting, quantified with Image J, and normalized to tubulin expression. Patients were classified into two groups by recursive partitioning method based on c-MYC protein expression in leukemic cells. The cells were grown in the absence and presence of NLC, and NLC dependency was determined as described in Methods. Association between c-MYC and NLC dependency was determined by χ² analysis. (E) BAFF is required for NLC to support the viability of high c-MYC expressors. Primary CLL cells with either high or low c-MYC expression determined by qPT-PCR and immunoblotting were cocultured with NLC with either a control Ig (IVIG) or a decoy BAFF receptor (BR3-FC fusion protein). Viability was determined by DIOC-6 and PI staining. Results (means ± SEM; n = 5) depict the ratio of viable cells observed in BR3-FC–treated cultures vs. IVIG-treated cultures.