Dimerization of MLL fusion proteins and FLT3 activation synergize to induce multiple-lineage leukemogenesis (original) (raw)
MLL-SEPT6 immortalizes murine hematopoietic progenitors with aberrant expression of Hox a7 and Hox a9 in vitro as MLL-ENL does. To examine the oncogenic potential of MLL-SEPT6 in vitro, MLL-SEPT6, the portion of MLL within MLL-SEPT6 (5′-MLL), SEPT6, and MLL-ENL short form (MLL-ENLs) (9) were subcloned into pMXs-neo (27) (Figure 1A). After confirmation, by Western blot analysis, of expression of these genes in packaging cells, the enriched murine hematopoietic progenitors were analyzed by the myeloid immortalization assay using retroviruses harboring these genes or harboring none of them (named mock) (Figure 1, B and C). Initial plating showed similar morphologies and reflected transduction efficiencies. In serial replating, MLL-SEPT6–transduced cells yielded and maintained increasing numbers of compact colonies similar to those generated by MLL-ENLs, although the cells transduced with mock, 5′-MLL, or SEPT6 rapidly failed to form colonies (Figure 1E). Wright-Giemsa–stained cytospin preparations of the cells constituting these compact colonies showed features consistent with myelomonocytic blasts (Figure 1F). Furthermore, RT-PCR of total RNA extracted from the colonies transduced with mock, MLL-SEPT6, and MLL-ENLs at the third round using primers Hoxa7S and Hoxa7AS, or Hoxa9S and Hoxa9AS, revealed that expression of both Hox a7 and Hox a9 was upregulated in MLL-SEPT6–transduced cells, as in MLL-ENLs (9), but not in mock-transduced cells (Figure 1G). The RT-PCR using primers M1S and M1AS also generated 468-bp PCR products as expected, and sequence analysis confirmed that these products represented transcripts of the Hox cofactor myeloid ecotropic viral integration site 1 (Meis1), which was reported to be upregulated with aberrant expression profiles of Hox (11). Unexpectedly, we could not detect any significant difference of expression levels of Meis1 among these colonies.
Immortalization of murine hematopoietic progenitors by MLL-SEPT6 fusion protein via aberrant expression of Hox genes. (A) Schematic representation of the retroviral constructions used. CXXC, CXXC domain; Zn fingers, zinc fingers; CS, cleavage sites; TAD, transactivation domain; SET, SET domain. (B) Western blot analysis of proteins extracted from PlatE cells transfected with the constructs shown in A, after immunoprecipitation using the anti-MLL Ab (lanes 1–9). Each lysate was blotted with the anti-FLAG Ab (lanes 1–9) or anti-SEPT6 Ab (lanes 10 and 11). Endogenous expression of SEPT6 was detected in lane 10. Lane 1, mock; lane 2, 5′-MLL; lane 3, MLL-SEPT6; lane 4, MLL-SEPT6Δcoil; lane 5, MLL-SEPT6Δcoil-ER; lane 6, MLL-SEPT6ΔGTP; lane 7, MLL-SEPT6ΔP-loop; lane 8, MLL-SEPT6S56N; lane 9, MLL-ENLs; lane 10, pMXs-neo alone (endogenous SEPT6); lane 11, SEPT6. (C) Experimental strategy for myeloid immortalization assay. (D) Myeloid immortalization assay using the constructs shown in A. Lanes for MLL-SEPT6Δcoil-ER indicate the presence (+) or absence (–) of 4-OHT. The bar graph shows numbers of colonies obtained after each round of replating in methylcellulose (average ± SD). (E and F) Typical morphology of the colonies generated by MLL-SEPT6 (E), and the cells constituting these colonies (F). Original magnification, ×40 (E), ×400 (F). (G) Expression of Hox a7, Hox a9, and Meis1 by RT-PCR in the cells from third-round cultures. β2m was used as an internal standard. M, 100-bp DNA ladder (New England Biolabs Inc.); lane 1, control (Ba/F3 with IL-3) cells; lane 2, mock; lane 3, MLL-SEPT6; lane 4, MLL-ENLs; lane 5, negative control.
Single-cell suspensions obtained from the third-round colony could grow and expand in liquid culture supplemented with IL-3 for more than 10 months but failed to survive without IL-3. These immortalized cells expressed Gr-1, CD11b, and c-kit but were negative for Sca-1, B220, CD3, and Ter119 as determined by fluorescence-activated cell sorting (FACS; BD Biosciences) analysis (Figure 2B and data not shown) and contained oligoclonal bands of proviral integration sites, as well as a single band of the full-length proviral DNA, by Southern blot analysis using the Neo probe (Figure 2, A and C). Expression of MLL-SEPT6 chimeric transcripts could be detected by RT-PCR of total RNA extracted from the immortalized cells (Figure 2D), while MLL-SEPT6 fusion proteins were hardly detectable in the lysate of the immortalized cells (data not shown). This showed the toxicity of high expression levels, as previously reported in the literature on leukemogenesis by MLL fusion proteins using retroviral transduction (9). Taken together, these results demonstrated that MLL-SEPT6 directly immortalizes murine hematopoietic cells via both the block of differentiation and the enhancement of self-renewal in vitro, with aberrant upregulation of Hox a7 and Hox a9, as MLL-ENL does.
Characterization of the cells immortalized by MLL-SEPT6. (A) Schematic representation of pMXs-neo-MLL-SEPT6. The restriction endonuclease sites and the Neo probe used in the Southern blot analysis are indicated by vertical arrows and a thick horizontal line, respectively. The primers used are indicated by horizontal arrows. LTR, long-terminal repeat. (B) Immunophenotype of the cells immortalized by MLL-SEPT6. Shadow profiles represent FACS staining obtained with Abs specific for the indicated cell surface antigens. Green lines represent staining obtained with isotype control Abs. (C) Southern blot analysis to detect clonality (lane 1) and integration (lane 2). Genomic DNA extracted from the cells immortalized by MLL-SEPT6 was digested with BamHI (lane 1) or NheI (lane 2), and hybridized with the Neo probe. Oligoclonal bands of proviral integration sites and a single band of the intact proviral DNA are indicated by arrows and an arrowhead, respectively. (D) Expression of MLL-SEPT6 fusion transcripts by RT-PCR. M, 1-kb DNA ladder (New England Biolabs Inc.); lane 1, cells immortalized by MLL-SEPT6; lane 2, negative control; lane 3, positive control (pMXs-neo-MLL-SEPT6).
Myeloid immortalization in vitro by MLL-SEPT6 requires dimerization of MLL fusion proteins through both its GTP-binding domain and its coiled-coil region. Because of common structural characteristics in 4 kinds of MLL-SEPTIN fusion proteins, we examined whether both a GTP-binding domain and a coiled-coil region were required for myeloid immortalization by MLL-SEPT6 in vitro, using various mutants shown in Figure 1A. Our myeloid immortalization assay demonstrated that lack of the GTP-binding domain (MLL-SEPT6ΔGTP), a highly conserved P-loop motif within the GTP-binding domain (MLL-SEPT6ΔP-loop), or the coiled-coil region (MLL-SEPT6Δcoil) led to failure in colony formation (Figure 1, B and D). Substitution of the critical single amino acid within the P-loop with reduction of GTP-binding activity (MLL-SEPT6S56N), based on previous findings of the well-characterized septin (17), was also found to lead to failure in maintenance of colonies (Figure 1D), and to result in no growth in the liquid culture, demonstrating that MLL-SEPT6S56N also failed to immortalize murine hematopoietic progenitors. These data indicated that the P-loop motif within the GTP-binding domain of SEPT6 was required for MLL-SEPT6–mediated immortalization, suggesting that GTP-binding activity was associated with MLL-SEPT6–mediated immortalization.
To examine whether MLL-SEPT6 homo-oligomerizes through its GTP-binding domain and/or coiled-coil region in the same way as other septins (18, 28), mutual-interaction assay of MLL-SEPT6 and its mutants using immunoprecipitation/Western blot analysis was first performed as shown in Figure 3. The intact MLL-SEPT6, not any mutants, was coprecipitated (Figure 3A), consistent with the activity of the immortalization in vitro. Furthermore, to investigate whether homo-oligomerization was sufficient for the immortalization, the coiled-coil region of pMXs-neo-MLL-SEPT6 was replaced with a mutant ligand-binding domain (LBD) of human estrogen receptor (hER) that induces dimerization specifically in response to 4-hydroxy-tamoxifen (4-OHT) (MLL-SEPT6Δcoil-ER) (29) (Figure 1A). Unexpectedly, MLL-SEPT6Δcoil-ER (Figure 1B) failed to induce the immortalization in the presence of 4-OHT (Figure 1D), suggesting that homo-oligomerization with proper whole structure is essential for MLL-SEPT6–mediated immortalization. On the other hand, immunofluorescent confocal microscopy of 293T cells transiently expressing MLL-SEPT6 or SEPT6 revealed that MLL-SEPT6 was distributed with a pattern of small speckles or dots in the nucleus (Figure 3B, bottom), while overexpression of SEPT6 alone did not shift its physiological localization in the cytoplasm (Figure 3B, top). Taken together, these data demonstrated that myeloid immortalization in vitro by MLL-SEPT6 requires homo-oligomerization in the nucleus, probably dimerization of MLL fusion proteins as reported (11, 12), through both its GTP-binding domain and its coiled-coil region.
Oligomerization of MLL-SEPT6 fusion protein through both its GTP-binding domain and its coiled-coil region in the nucleus. 293T cells were cotransfected with equal amounts of FLAG-tagged and HA-tagged constructs (A), and transfected with pMXs-neo-SEPT6 or pMXs-neo-MLL-SEPT6 (B). (A) Self-interaction among MLL-SEPT6 fusion proteins or MLL-SEPT6 mutants, analyzed by Western blot analysis after immunoprecipitation. Lysates of 293T cells coexpressing FLAG-tagged and HA-tagged MLL-SEPT6 or its mutants (top and middle) were immunoprecipitated by the anti-FLAG Ab, and lysates of the cells expressing HA-tagged MLL-SEPT6 or its mutants (bottom) were immunoprecipitated by the anti-HA Ab. Anti-FLAG immunoprecipitates were blotted with the anti-HA Ab (top) or the anti-FLAG Ab (middle), while anti-HA immunoprecipitates were blotted with the anti-HA Ab (bottom). Lane 1, MLL-SEPT6; lane 2, MLL-SEPT6Δcoil; lane 3, MLL-SEPT6ΔP-loop; lane 4, MLL-SEPT6S56N. (B) Localization of SEPT6 and MLL-SEPT6, analyzed by immunofluorescent confocal microscopy. FITC-conjugated secondary Abs reacting with the anti-FLAG Ab in 293T cells expressing SEPT6 or MLL-SEPT6 visualized their cellular localizations (middle). Nuclei were visualized with DAPI (left), and merged images are displayed (right). Original magnification, ×400.
MLL-SEPT6 induces lethal MPD, not acute leukemia, in vivo. We next examined the leukemogenic potential of MLL-SEPT6 in vivo using transplantation of the cells immortalized by MLL-SEPT6 into sublethally irradiated syngeneic mice. Five of 8 mice transplanted with the immortalized cells died with latencies of over 6 months within an observation period of 15 months (Table 1). Morbid mice that received the transplantation exhibited hepatosplenomegaly with leukocytosis, anemia, and thrombocytopenia (Table 1). Notably, histopathologic analysis showed that bone marrow and peripheral blood cells derived from the morbid mice had morphologic and immunophenotypic features of MPD with myeloid hyperplasia consisting predominantly of mature granulocytic elements on FACS analysis, and that these elements infiltrated the spleen and the liver (data not shown). The lethal MPD was also induced by transplantation of murine hematopoietic progenitors transduced with MLL-SEPT6 into lethally irradiated syngeneic mice directly after retroviral infection (described in detail below), and bone marrow cells harvested from these MPD mice grew and expanded dependently on IL-3 in vitro (data not shown). These results differ from findings in the previous reports in which acute leukemias with latencies of 2–6 months were induced by some MLL fusion proteins (9, 10), which suggested that fusion of MLL with SEPT6 was not sufficient, and required additional genotoxic stress, to induce acute leukemia.
Characteristics of morbid mice transplanted with cells immortalized by MLL-SEPT6 in vitro and mice transplanted with hematopoietic progenitors, directly after transduction with MLL-SEPT6, MLL-ENLs, and/or FLT3-ITD
Secondary genotoxic stress, such as FLT3-ITD, synergistically transforms hematopoietic progenitors transduced with MLL-SEPT6 or MLL-ENL in vitro. To seek a candidate for additional genotoxic stress required for leukemogenesis by MLL-SEPT6, we first analyzed the transforming activity of _MLL-SEPT6_–transduced cells with additional expression of _FLT3_-ITD, using the transformation assay (Figure 4, A and C). The infection efficiencies of _FLT3_-ITD and vector alone were 0.77% ± 0.31% and 9.0% ± 0.67%, respectively. The transduction of _FLT3_-ITD not only enabled the immortalized cells to grow without IL-3 but also turned almost all of the cells (95.0% ± 1.5%) positive for GFP 7 days after the deprivation of IL-3, while transduction of the vector alone did not (Figure 4B). These results suggest that _FLT3_-ITD has the potential to replace the signaling pathways by IL-3 in the cells expressing MLL-SEPT6 in vitro. The transduction of a kinase-active mutant FLT3 with an Asp-835-to-Val mutation (FLT3D835V) also transformed the immortalized cells (data not shown) in the same transformation assay, as it has been reported to transform Ba/F3 cells (30).
Synergistic transformation of murine hemato-poietic progenitors by MLL fusion genes and FLT3-ITD in vitro. (A) Schematic representation of the retroviral constructions expressing FLT3-ITD. (B) Transformation assay of the cells immortalized by MLL-SEPT6, after transduction with FLT3-ITD in the pMY-IRES-EGFP construct shown in A in the presence (+) or absence (–) of IL-3. (C) Western blot analysis of proteins extracted from PlatE cells transfected with the constructs shown in A and each vector alone as a control, after immunoprecipitation using the anti-FLT3 Ab (lanes 1–4). Each lysate was blotted with the anti-FLT3 Ab. Lane 1, pMY-IRES-EGFP alone; lane 2, pMY-FLT3-ITD-IRES-EGFP; lane 3, pMYpuro alone; lane 4, pMYpuro-FLT3-ITD. (D) Myeloid immortalization assay using the pMYpuro constructs shown in A. The bar graph shows numbers of colonies obtained after each round of replating in methylcellulose (average ± SD). (E) Myeloid transformation assay using the sequential transduction with FLT3-ITD or control (pMYpuro alone) after MLL-SEPT6, MLL-ENLs, or mock (pMXs-neo alone) in the presence (+) or absence (–) of IL-3.
On the other hand, the myeloid immortalization assay using _FLT3_-ITD in pMYpuro could not detect any myeloid immortalization (Figure 4, A, C, and D), which implies that _FLT3_-ITD alone was insufficient for immortalization of murine hematopoietic progenitors in this colony assay or that this assay might be inappropriate to examine the oncogenic proliferative and/or survival advantage, which was considered to be primarily conferred by some oncogenic mutations including _FLT3_-ITD (31). Furthermore, the synergy of MLL-SEPT6 and _FLT3_-ITD was examined with myeloid transformation assay using either simultaneous or sequential transduction with these genes. To generalize the synergy in the _MLL_-mediated transformation in vitro, _MLL-ENL_s was also applied to the same assay. While the simultaneous transduction failed to cause transformation, probably because of the low efficiency of double transduction and limitation of the sensitivity in this in vitro assay (data not shown), the sequential transduction with _FLT3_-ITD after MLL-SEPT6 or _MLL-ENL_s, not mock, enabled murine hematopoietic progenitors to grow and expand without IL-3 (Figure 4E). Taken together, these results demonstrated that MLL fusion genes MLL-SEPT6 and MLL-ENL can transform murine hematopoietic progenitors in vitro in concert with additional genotoxic stress, such as _FLT3_-ITD, which suggests that MLL fusion gene and _FLT3_-ITD may synergistically serve as oncogenes through different pathways.
MLL fusion genes MLL-SEPT6 and MLL-ENL require secondary genotoxic stress, such as FLT3-ITD, to induce acute leukemias in vivo with short latency. To address the synergistic leukemogenic potential of MLL fusion gene and _FLT3_-ITD in vivo, we directly transplanted Ly-5.1 murine hematopoietic progenitors transduced with MLL-SEPT6 and _FLT3_-ITD (MS6/FLT3) using a combination of each retrovirus into lethally irradiated syngeneic Ly-5.2 mice. The other combinations of each retrovirus — mock/GFP (pMXs-neo and pMY–internal ribosomal entry site–enhanced GFP [pMY-IRES-EGFP]), MS6/GFP, and mock/FLT3 — were also given as controls. In contrast to the MS6/GFP mice, which died with latencies of 57–147 days (120 ± 32 days), the MS6/FLT3 mice died with significantly shorter latencies of 32–91 days (63 ± 18 days, P < 0.05, log-rank test) (Figure 5A and Table 1). The morbid MS6/FLT3 mice exhibited mild hepatomegaly and moderate splenomegaly with various ranges of leukocytosis, anemia, and thrombocytopenia (Table 1). Some of the morbid MS6/FLT3 mice also exhibited remarkable lymphadenopathy (Figure 5C). Histopathologic analysis showed that the majority of bone marrow or peripheral blood cells derived from the morbid MS6/FLT3 mice had morphologic features of immature myelomonocytic blasts, which severely infiltrated the spleen and the liver (Figure 5C). The morphology of the differentiation blockade suggested that the MS6/FLT3 mice developed acute leukemias. On the other hand, the MS6/GFP mice died of MPD that showed features (Figure 5, A and C, and Table 1) similar to those seen in mice transplanted with the cells immortalized by MLL-SEPT6 in vitro, while the mock/FLT3 mice survived without hematological disorders in peripheral blood for an observation period of 6 months (Figure 5A and Table 1). Histopathologic analysis of 1 mock/FLT3 mouse sacrificed 160 days after the transplantation demonstrated neither significant hepatosplenomegaly nor any disorders in the bone marrow, where 20% of the cells were positive for GFP and hence expressed _FLT3_-ITD (data not shown).
Synergistic leukemogenesis induced by MLL fusion genes and FLT3-ITD in vivo. (A and B) Survival curves of mice transplanted with mock/GFP (n = 4), MLL-SEPT6/GFP (MS6/GFP; n = 6), mock/FLT3 (n = 5), and MS6/FLT3 (n = 9) (A), and with MLL-ENLs/GFP (MEs/GFP; n = 4) and MEs/FLT3 (n = 5) (B). Mice transplanted with MLL fusion genes in combination with FLT3-ITD showed significantly shorter survival time than those with corresponding MLL fusion genes in combination with GFP (P < 0.05, log-rank test). (C) Representative macroscopic and histopathologic analysis of morbid mice transplanted with MS6/FLT3 and MS6/GFP, respectively. Arrowheads show lymphadenopathy. BM cells were stained with Wright-Giemsa, and paraffin sections of liver and spleen were stained with H&E. Original magnification of BM cells, ×200; scale bars, 200 μm.
Cytological analysis showed that almost all of the bone marrow cells (94.4% ± 9.0%) derived from the MS6/FLT3 mice originated in donor cells (Ly-5.1), and that the majority of these cells (85.4% ± 14.9%) were positive for GFP and thus expressed _FLT3_-ITD (Figure 6). Southern blot analysis of DNA derived from the spleen of the MS6/FLT3 mice demonstrated that intensities of proviral integration bands of MLL-SEPT6 and _FLT3_-ITD were almost equal, and that oligoclonal bands of proviral integration sites were also present (Figure 7, A and B). In addition, expression of MLL-SEPT6 was detected by RT-PCR analysis of total RNA derived from the spleens (data not shown), indicating both cointegration and coexpression of MLL-SEPT6 and _FLT3_-ITD in the leukemic cells. Immunophenotyping analysis revealed that these leukemic cells were highly positive for Gr-1 and CD11b (Figure 6). Interestingly, the leukemic cells with these myeloid markers sometimes coexpressed B220, but no CD3 (Figure 6 and data not shown), and B220 expression did not always correlate with IgH gene rearrangements (Figure 7C). For example, 57.0% and 98.2% of the GFP-positive leukemic cells derived from mouse no. 213 were positive for B220 and CD11b, respectively (Figure 6), which demonstrates that at least half of these leukemic cells were biphenotypic, although more direct evidence from 3- or 4-color flow cytometry analysis could not be obtained because of lack of these leukemic cells. These results suggest that MLL-SEPT6 and _FLT3_-ITD could induce acute myeloid, or sometimes biphenotypic, leukemia with lineage promiscuity (32). Peroxidase stain of these leukemic cells carrying B220 also corroborated myeloid lineage of these blasts (data not shown).
Immunophenotype of bone marrow cells obtained from representative mice (nos. 213 and 212) transplanted with mock/GFP, MS6/GFP, and MS6/FLT3. The dot plots show each surface antigen labeled with a corresponding PE-conjugated mAb versus expression of GFP.
Southern blot analysis of spleen DNA obtained from representative mice transplanted with mock/GFP, MS6/GFP, and MS6/FLT3. Genomic DNA extracted from each spleen was digested with NheI (A), BamHI (B), or EcoRI (C) and hybridized with the Neo probe (A and B, left), the GFP probe (A and B, right), or the JH probe (C). (A) Each single band of intact proviral DNA, including MLL-SEPT6 (MS6) and FLT3-ITD (FLT3), is indicated by an arrow with each abbreviation. (B) Oligoclonal bands of proviral integration sites of MS6 or FLT3 are indicated by black or white arrowheads, respectively. (C) Germ line (G) or rearrangements (R) of IgH gene are indicated by an arrow with each abbreviation. Lane 1, mock/GFP mouse; lanes 2 and 3, MS6/GFP mice; lanes 4 and 5, MS6/FLT3 mice.
To generalize the synergetic effect in vivo in _MLL_-mediated leukemogenesis, _MLL-ENL_s was also applied to this leukemogenesis assay. _MLL-ENL_s and _FLT3_-ITD (MEs/FLT3) could induce AML with significantly shorter latency of 17.6 ± 1.8 days (P < 0.05, log-rank test), while _MLL-ENL_s (MEs/GFP) induced MPD with latency of 92 ± 9.1 days (Table 1, Figure 5B, and data not shown) as previously described (14). Taken together, these data demonstrate that MLL fusion genes MLL-SEPT6 and MLL-ENL can synergize with secondary genotoxic stress such as _FLT3_-ITD to induce acute myeloid or biphenotypic leukemias in vivo with short latency as a clinical feature.