B cell activator PAX5 promotes lymphomagenesis through stimulation of B cell receptor signaling

Abstract

The presumed involvement of paired box gene 5 (PAX5) in B-lymphomagenesis is based largely on the discovery of Pax5-specific translocations and somatic hypermutations in non-Hodgkin lymphomas. Yet mechanistically, the contribution of Pax5 to neoplastic growth remains undeciphered. Here we used 2 Myc-induced mouse B lymphoma cell lines, Myc5-M5 and Myc5-M12, which spontaneously silence Pax5. Reconstitution of these cells with Pax5-tamoxifen receptor fusion protein (Pax5ER(TAM)) increased neoplastic growth in a hormone-dependent manner. Conversely, expression of dominant-negative Pax5 in murine lymphomas and Pax5 knockdown in human lymphomas negatively affected cell expansion. Expression profiling revealed that Pax5 was required to maintain mRNA levels of several crucial components of B cell receptor (BCR) signaling, including CD79a, a protein with the immunoreceptor tyrosine-based activation motif (ITAM). In contrast, expression of 2 known ITAM antagonists, CD22 and PIR-B, was suppressed. The key role of BCR/ITAM signaling in Pax5-dependent lymphomagenesis was corroborated in Syk, an ITAM-associated tyrosine kinase. Moreover, we observed consistent expression of phosphorylated BLNK, an activated BCR adaptor protein, in human B cell lymphomas. Thus, stimulation of neoplastic growth by Pax5 occurs through BCR and is sensitive to genetic and pharmacological inhibitors of this pathway.

Figure 1. Contribution of Pax5 to neoplastic growth.

(A) Immunoblotting demonstrating reactivation of Pax5 in 2 independent Myc5 tumors obtained from short-term cultures (T1 and T2) and lack of such reactivation in Myc5 tumors derived from long-term cultures (M5 and M12). Myc5 cultures engineered to overexpress Pax5 (Ctrl) were used as a positive control. (B) Myc5-M5 cells transduced with the Pax5 retrovirus maintain Pax5 expression in vivo (T5–T8). Vector-transduced cells were used as a negative control (T1–T4). (C) Pax5-transduced Myc5-M5 cells form more rapidly growing tumors than vector-transduced cells. Plotted on the y axis are average slope values. Error bars denote SD. In this and subsequent tumor load experiments, no less than 7 mice were used for each treatment group. (D) Flow cytometric analysis of representative tumors from C. Control tumors (left) were positive for the myeloid marker CD11b and negative for the B cell marker CD19. Pax5-transduced tumors (right) contain both CD11b^–CD19^– and CD11b^–CD19⁺ cells. (E) Staining of formalin-fixed, paraffin-embedded sections with an antibody against B cell marker CD45R. Vector-transduced control tumors did not contain CD45R⁺ cells. Some Pax5-transduced tumors contain islets of CD45R⁺ cells (blue stain). Normal spleen was used as a positive control. Original magnification, ×4 (insets, ×20).

Figure 2. Growth of tumors with conditionally active Pax5.

(A) Reconstitution of Myc5-M5 cells with Pax5ER. Immunoblotting on transduced cell lysates was performed using an anti-Pax5 antibody. (B) Enhanced nuclear localization of Pax5ER following treatment with 4OHT. Right panels show immunocytochemical staining with an anti-Pax5 antibody. Left panels show counterstaining of nuclei with DAPI. (C) Kinetics of tumor growth by Pax5ER and control (GFP only) cells in animals treated with 4OHT or vehicle only. (D) Kinetics of tumor growth by short-term Myc5 cultures expressing either Pax5 or its dominant-negative mutant (Pax5DN) lacking the activation domain. The small increase in tumor size conferred by Pax5 expression was not statistically significant; the decrease in tumor sizes conferred by overexpression of the dominant-negative Pax5 mutant was significant.

Figure 3. Pax5 affects expression of BCR signaling components.

(A) Mapping of Pax5 target genes to the BCR pathway (http://www.genome.jp/dbget-bin/www_bget?path:mmu04662). Triple arrows pointing up and down denote Pax5-activated and -repressed genes, respectively. DAG, diacylglycerol, which is produced by PLCγ2 and directly activates PKCβ; +p, phosphorylation; –p, dephosphorylation. (B) Immunoblotting using an anti-CD19 antibody. The 2 bands most likely correspond to the reduced and unreduced forms of the protein (http://mpr.nci.nih.gov/PROW/guide/1916589419_g.htm). Three independent tumors from each group were tested. All tumors were from 4OHT-treated animals. (C) Flow cytometric detection of CD22 in GFP-only and Pax5-expressing Myc5-M5 tumors. Gray and black plots denote anti-CD22–stained and control cells (not stained with the primary antibody), respectively. (D) Real-time RT-PCR analysis of tumors from Figure 2C. Three independent tumors from each group were used for analysis, and each cDNA was tested in triplicate. Values on the y axis equal 2^[30–Ct]. Error bars denote SD. In case of CD79a (Igα), significant upregulation was observed in Pax5ER TAM cells even in the absence of 4OHT. All genes shown are Pax5 with the exception of Pirb, a Pax5-repressed gene. All values are normalized for β-actin.

Figure 4. Role of BCR signaling in Pax5-dependent neoplastic growth.

(A) Kinetics of tumor growth by Myc5-M5 cells expressing GFP alone, Pax5, MAHB, or the inactive form of MAHB (MAHBmut). Both Pax5 and MAHB significantly promoted tumorigenesis. (B) Immunoblotting detecting ectopic expression of Pax5, MAHB, and MAHBmut in representative tumors from A. (C) Flow cytometric measurement of CD22 levels in Myc5-M5–Pax5ER cells transduced by the empty vector or the CD22-encoding retrovirus. (D) Kinetics of tumor growth by cells from C in animals treated with 4OHT or vehicle alone. (E) Kinetics of tumor growth by Myc5-M5–Pax5ER cells in animals continuously treated or treated with and subsequently deprived of 4OHT (with,w/o 4OHT). Arrow indicates the day when animals were assigned to different treatment groups. The difference in tumor size between the 2 groups became significant 1 day after randomization. (F) Kinetics of tumor growth by Myc5-M5–Pax5ER cells in animals continuously treated with 4OHT, treated with and subsequently deprived of 4OHT, or treated with 4OHT and Syk inhibitor (SykIn). Arrow indicates the day when animals were assigned to different treatment groups. The difference in tumor size between the 4OHT and 4OHT plus Syk inhibitor groups became significant 2 days after randomization.

Figure 5. In vitro effects of Pax5 and CD22.

(A) Cell accumulation in untreated and 4OHT-treated cultures of Pax5ER and GFP-only Myc5-M5 cells. Cells were counted in triplicate; error bars denote SD. Insets represent ³H-thymidine incorporation assay performed on the same cultures. (B) Cell accumulation in untreated and 4OHT-treated cultures of CD22- and vector-transduced Myc5-Myc5–Pax5ER cells. (C) Pax5 knockdown in human SUDHL-4 and SUDHL-6 DLBCL lines using lentivirally encoded shRNAs. Five different hairpins were first tested on SUDHL-4 cells. The most effective shRNA, 6061, was also introduced into SUDHL-6 cells. Pax5 and actin levels were determined using immunoblotting. (D) Cell accumulation assay following Pax5 knockdown. Cells transduced by shRNA 6061 and control virus were seeded at equal densities and counted 48 hours later. P values were determined using unpaired Student’s t test.

Figure 6. Immunohistochemical analysis of BLNK phosphorylation in human B cell neoplasms.

Archival formalin-fixed, paraffin-embedded sections of 2 diffuse large B cell lymphomas (tumors A and B reflect positive and negative samples, respectively) and 1 multiple myeloma were stained with antibodies against Pax5 and phosphorylated BLNK (pBLNK) as described in Methods. Brown staining indicates positivity for proteins tested. Blue staining depicts counterstained nuclei. Original magnification, ×20.