MLL-ENL cooperates with SCF to transform primary avian multipotent cells

Abstract

The MLL gene is targeted by chromosomal translocations, which give rise to heterologous MLL fusion proteins and are associated with distinct types of acute lymphoid and myeloid leukaemia. To determine how MLL fusion proteins alter the proliferation and/or differentiation of primary haematopoietic progenitors, we introduced the MLL-AF9 and MLL-ENL fusion proteins into primary chicken bone marrow cells. Both fusion proteins caused the sustained outgrowth of immature haematopoietic cells, which was strictly dependent on stem cell factor (SCF). The renewing cells have a long in vitro lifespan exceeding the Hayflick limit of avian cells. Analysis of clonal cultures identified the renewing cells as immature, multipotent progenitors, expressing erythroid, myeloid, lymphoid and stem cell surface markers. Employing a two-step commitment/differentiation protocol involving the controlled withdrawal of SCF, the MLL-ENL-transformed progenitors could be induced to terminal erythroid or myeloid differentiation. Finally, in cooperation with the weakly leukaemogenic receptor tyrosine kinase v-Sea, the MLL-ENL fusion protein gave rise to multilineage leukaemia in chicks, suggesting that other activated, receptor tyrosine kinases can substitute for ligand-activated c-Kit in vivo.

Fig. 1. Growth of MLL–AF9- and MLL–ENL-infected cultures. (A) MLL–AF9- (solid circles) and MLL–ENL (open squares)-infected mass cultures, as well as control cultures infected with empty vector (+ and ×), were cultivated in multipotent cell medium and cumulative cell numbers determined daily by counting in an electronic counter (see Materials and methods). (B) Four clones verified for single MLL–ENL integration sites: D8 (triangles), D11 (diamonds), B8 (circles) and IIC10 (squares) were cultured in multipotent cell medium containing (+SCF) or lacking (–SCF) SCF. Cumulative cell numbers were determined as above.

Fig. 2. Molecular analysis of MLL–ENL-infected cultures. (A) Schematic representation of the CRNCM-MLL–ENL retroviral vector used in this study. (B) The presence of full-length MLL–ENL cDNA in nine multipotential methocel clones was detected by Southern blot analysis, digesting genomic DNA with EcoRI and hybridizing with a human MLL–ENL probe (1.25 kb BamHI–BstXI fragment, see A). (C and D) Monoclonal origin of MLL–ENL-infected clonal cultures. To identify clones containing single retroviral integration sites, genomic DNA from nine clones grown in multipotential medium (C) and from four clones after terminal differentiation (D) into myeloid (left panel) or erythroid cells (right panel; see Figure 4 and Materials and methods) was subjected to Southern blot analysis after digestion with BamHI and hybridization with the same human MLL–ENL probe as in (B). Genomic DNA from the chicken T-cell line MSB1 was used as control for cross-reactive endogenous chicken MLL.

Fig. 3. MLL–ENL-infected cultures express multilineage cell surface markers. Cell surface marker profiles for MLL–ENL clones IIC10 and D11 as determined by FACS analysis. For a detailed description of antigens detected by the panel of antibodies used, see Results.

Fig. 4. MLL–ENL-transformed multipotent clones can be induced to differentiate along erythroid and myeloid lineages. Cells from MLL–ENL clones B8 (top panels) and IIC10 (bottom panels) were cytocentrifuged on to slides after cultivation in multipotent cell medium (left panels) or after induction of terminal erythroid (middle panels) or myeloid differentiation (right panels), employing a two-step commitment/differentiation protocol (Beug et al., 1995a; Materials and methods). Photographs of representative cytospins are shown after staining with acid benzidine (haemoglobin, yellow-brown) plus Wright–Giemsa (Beug et al., 1995b). Insets: cells at higher magnification.

Fig. 5. Molecular and phenotypic analysis of MLL–ENL/ts-v-Sea- induced leukaemias. (A) Southern blot of genomic DNA extracted from bone marrow of leukaemic chickens. Digestion with EcoRI reveals a truncated MLL–ENL cDNA of ∼4 kb in all leukaemias analysed (see legend to Figure 2). As a control, the MLL–ENL clone D11 containing the 6 kb full-length MLL–ENL cDNA and the chicken T-cell line MSB1 are shown. (B) Schematic representation of MLL–ENL-specific sequences detected in leukaemias 2 and 6, revealing an in-frame deletion. See Results and Supplementary data for a detailed description of the respective nested PCR analyses. (C) Analysis of ts-v-Sea in leukaemias 2, 6 and 7 by PCR analysis generated the expected 1.4 kb product, encompassing the S13 viral env sequence plus the transmembrane and complete tyrosine kinase domains of v-Sea. Positive control, in vitro-transformed ts-v-Sea/MLL–ENL clone; negative control, MLL–ENL clone D8; W, water control. (D) Cultured cells from leukaemia 2 (two fields, top) and leukaemia 6 (bottom) are shown after cytocentrifugation on to slides and staining with acid benzidine and Wright–Giemsa.

Fig. 6. ts-v-Sea can substitute for SCF-induced c-Kit signalling required for leukaemic transformation by MLL–ENL. In vitro-transformed MLL–ENL/ts-v-Sea clones C7, D6 and G8 were cultivated in S13 medium without additions at 37°C (triangles) or at 42°C in the same medium plus 300 µM of the glycosylation inhibitor castanospermine, in the presence (squares) or absence (circles) of SCF. Cumulative cell numbers were determined daily using an electronic cell counter.