Oscillation between B-lymphoid and myeloid lineages in Myc-induced hematopoietic tumors following spontaneous silencing/reactivation of the EBF/Pax5 pathway

Abstract

B lymphomagenesis is an uncontrolled expansion of immature precursors that fail to complete their differentiation program. This failure could be at least partly explained by inappropriate expression of several oncogenic transcription factors, such as Pax5 and Myc. Both Pax5 and c-Myc are implicated in the pathogenesis of non-Hodgkin lymphomas. To address their role in lymphomagenesis, we analyzed B-cell lymphomas derived from p53-null bone marrow progenitors infected in vivo by a Myc-encoding retrovirus. All Myc-induced lymphomas invariably maintained expression of Pax5, which is thought to be incompatible with terminal differentiation. However, upon culturing in vitro, several cell lines spontaneously down-regulated Pax5 and its target genes CD19, N-Myc, and MB1. Unexpectedly, other B-cell markers (eg, CD45R) were also down-regulated, and markers of myeloid lineage (CD11b and F4/80 antigen) were acquired instead. Moreover, cells assumed the morphology reminiscent of myeloid cells. A pool of F4/80-positive cells as well as several single-cell clones were obtained and reinjected into syngeneic mice. Remarkably, pooled cells rapidly re-expressed Pax5 and formed tumors of relatively mature lymphoid phenotype, with surface immunoglobulins being abundantly expressed. Approximately half of tumorigenic single-cell clones also abandoned myeloid differentiation and gave rise to B lymphomas. However, when secondary lymphoma cells were returned to in vitro conditions, they once again switched to myeloid differentiation. This process could be curbed via enforced expression of retrovirally encoded Pax5. Our data demonstrate that some Myc target cells are bipotent B-lymphoid/myeloid progenitors with the astonishing capacity to undergo successive rounds of lineage switching.

Figure 1. Myc5 B-lymphoma cells acquire myeloid phenotype in vitro

(A) Histolopathologic (left) and cytologic (right) analyses of Myc3 and Myc5 lymphomas maintained in vivo and in vitro, respectively. On the left, sections of tumor tissues were stained with H&E (original magnification, × 40). On the right, cultured cells were spun onto slides and stained with Wright-Giemsa (Hemo-Quick, original magnification, × 60). (B) Flow cytometric analyses of surface markers on primary (in vivo) and cultured (in vitro) Myc3 and Myc5 cells. Cells stained with specified antibodies are indicated by black histograms; control unstained cells, with open lines. + and − refer to positive and negative staining, respectively.

Figure 2. Cultured myeloid Myc5 cells reacquire B-lymphoma markers in vivo

(A) PCR analysis confirming the presence of VDJ-rearrangement in cultured Myc5 cells (far right lane). In preceding lanes, DNAs from primary Myc5 tumor and murine fibroblasts were used as positive and negative controls, respectively. Migration of the rearranged fragment is indicated by the arrow. (B) FACS of cultured Myc5 cells into B220-positive (R1), F4/80-positive (R3), and doubly positive (R2) populations. As indicated by the arrow, the R3 population was expanded and used for tumor production. (C) Flow cytometric analyses of surface markers on sorted Myc5 cells from the R3 fraction that were expanded in culture (in vitro) and subsequently injected into animals and allowed to form secondary tumors (in vivo). Cells stained with specified antibodies are indicated by black histograms; control unstained cells, with open lines. + and − refer to positive and negative staining, respectively. (D) The left panel depicts the same analyses as in panel C but performed on single cell subclones 5/12 and 5/5 from the R3 fraction. The right panel refers to H&E staining of sections of tumors derived from clones 5/12 and 5/5. Original magnification × 40.

Figure 3. Expression of B-specific transcription factors and their targets is altered in some Myc-tumor cell lines

All panels depict RT-PCR analyses of mRNAs encoding proteins specified on the left. Primary (1°) tumors, cultured cells, or secondary tumors derived from cultured cells (2°) were used as sources of RNA. +/− RT refers to reactions carried out in the presence and absence of reverse transcriptase. B16.F10 murine melanoma cells were used as a negative control. Dotted box indicates lack of expression of EBF, Pax5, and Pax5 target genes. Expression of the Pax5 protein in primary tumors was also confirmed using Western blotting (data not shown).

Figure 4. Ectopic expression of Pax5 prevents acquisition of myeloid markers by Myc5 B-lymphoma cells

(A) The diagram of the Pax5-encoding retrovirus. The GFP-encoding MIGR1 retroviral vector was used as a backbone. (B) FACS of Pax5- or control retrovirus-infected Myc5 cells from a secondary tumor. Sorted populations are indicated by rectangles and arrows. (C) Flow cytometric analyses of the myeloid CD11b surface marker on Myc5 cells infected by the control (top panels) and Pax5-encoding (bottom panels) retroviruses.