Conditional expression of Ott-MAL in a knockin mouse model.
We engineered a conditional Ott-MAL knockin mouse model that allowed for expression of the fusion protein from the endogenous murine Ott promoter after Cre recombinase–mediated excision of a STOP cassette flanked by loxP recombination sites (floxed-STOP; Figure A). Southern blot analysis of XmnI-digested ES cell DNA identified clones with homologous recombination (Figure , B and C). Functional integrity of the targeted construct was confirmed by transducing Ott3lox/WT ES clones with adenovirus encoding the Cre recombinase, followed by selection of OttOtt–MAL excised clones based on Southern blot analysis (Figure D). Conditional expression of the fusion mRNA and protein was confirmed by RT-PCR and Western blot analysis, respectively (Figure , E and F). Animals with germline transmission of the Ott3lox allele were crossed to an EIIA-Cre transgenic mouse line, resulting in in vivo excision of the floxed-STOP cassette, with germline transmission of the OttOtt–MAL allele. OttOtt–MAL/WT animals (termed OM) were observed at predicted Mendelian ratios and were backcrossed into the C57BL/6 background for further analysis.
OTT-MAL expression results in abnormal fetal and adult hematopoiesis but induces AMKL with low penetrance.
As the OTT-MAL fusion is restricted to infant leukemias and is likely to occur in utero, we first analyzed fetal liver hematopoiesis using flow cytometry. Plating of purified Lineage–Sca1+c-Kit+ (LSK) cells from E12.5 fetal livers revealed a significant increase in colony-forming efficiency due to expansion of multipotent myeloid progenitors (CFU–granulocyte-macrophage-erythroid-Mk [CFU-GEMM]) in OM versus WT cells (Figure A), although no differences were observed in the absolute number of hematopoietic progenitors or CD41+ cells between OM and WT fetal liver at day E12.5 (Supplemental Figure 1; supplemental material available online with this article; doi:10.1172/JCI35901DS1; and data not shown). Furthermore, replating of LSK cell–derived primary colonies from OM embryos showed an increase in both Mk-containing (CFU-GEMM) and pure Mk (CFU-Mk) secondary colonies (Figure B), whereas no CFU-Mk were obtained with plating of WT cells. Identification of Mk-containing colonies was confirmed by staining cytospins of single colonies for acetylcholinesterase (AchE) activity (data not shown). These data indicate that OTT-MAL expression results in aberrant differentiation of HSC toward the megakaryocytic lineage in vitro.
OTT-MAL induces abnormal fetal and adult hematopoiesis and AMKL with low penetrance.
We next investigated the effect of OTT-MAL expression on adult hematopoiesis. As OM animals did not show hematological abnormalities during the first 6 months of life (data not shown), we studied an older cohort of 15 OM, 10 Ott3lox/WT, and 10 WT littermate animals aged 18–24 months. Since extramedullary hematopoiesis leading to splenomegaly is a hallmark of AMKL in humans, we first evaluated the number of progenitors in splenocytes and observed an increase in OM animals (Figure C). In addition, these cells also showed enhanced replating activity in the majority of OM animals (8/15), whereas no replating activity was observed in cells derived from WT littermate (0/10) or Ott3lox animals (0/10) (Figure C). Colonies derived from OM animals at the fifth round of replating showed AchE+ cells, indicating their megakaryocytic nature (data not shown). In addition, histopathologic analysis revealed that animals with increased replating activity of splenocytes had abnormalities in the hematopoietic system including extramedullary hematopoiesis in the spleen and liver (6/15) and frank leukemia (2/15: nos. 6133 and 8904). These leukemias had phenotypic attributes of human AMKL, including infiltration of the BM, spleen, liver, and kidney with an admixture of immature megakaryocytic and erythroid elements as well as megakaryoblasts circulating in the peripheral blood (Supplemental Figure 2). The disease was transplantable into secondary and tertiary recipients, with a median survival of 45 days and 24 days, respectively (Figure D). Secondary recipients showed a phenotype similar to that of primary animals, with infiltrating leukemic blasts that were vWF+, confirming the megakaryoblastic nature of the disease (Figure E and Supplemental Figure 3).
A cytokine-dependent cell line was derived from leukemic cells of animal no. 6133 (Figure F). Similar to primary leukemic cells from this animal, the 6133 cell line displayed an immature phenotype (Figure G). These cells were positive for c-Kit as well as several megakaryocytic markers, including CD41 and CD42b, and were negative for myeloid markers, including Mac1 and FcεRIa (Figure H). Together, these results indicate that expression of OTT-MAL results in abnormal differentiation of fetal hematopoietic progenitors, enhanced self-renewal properties of MkPs, and extramedullary hematopoiesis, leading to development of AMKL with low penetrance and long latency.
OTT-MAL activates RBPJ-mediated transcriptional activity.
OTT belongs to a family of proteins that interact with the transcription factor RBPJ (11
), and constitutive activation of RBPJ-mediated transcription due to Notch signaling has been reported to result in increased self renewal and cancer (25
). To better understand the mechanism of megakaryocytic transformation by OTT-MAL, we determined whether OTT-MAL could interfere with RBPJ-mediated transcriptional regulation. Coimmunoprecipitation experiments in 293T cells showed that OTT-MAL formed a complex with RBPJ that required the RNA recognition motif (RRM) domains of OTT-MAL (Figure , A and B). Furthermore, transactivation assays in 293T cells using an RBPJ luciferase reporter showed that OTT-MAL activated RBPJ-mediated transcription in a dose-dependent manner, whereas expression of OTT resulted in a subtle inhibition of RBPJ transcription (Figure C) (13
). Transactivation of the RBPJ reporter by OTT-MAL was dependent on the transactivation domain (TAD) of MAL and the RRM domains of OTT (Figure , B and C).
OTT-MAL activates RBPJ-mediated transcriptional activity.
To investigate whether the RBPJ target genes could be transcriptionally activated by OTT-MAL in vivo, we first crossed OM animals with an established transgenic Notch reporter (TNR) mouse model, in which the expression of a GFP reporter is under the control of RBPJ response elements (27
). GFP fluorescence was higher in lineage-negative hematopoietic cells from double OM+TNR animals than in cells from control TNR animals (Figure D). In addition, primary lineage negative hematopoietic cells from OM animals showed increased expression of Hes1, Hes5, and Dtx1 transcripts compared with WT animals (Figure E), confirming activation of RBPJ transcription by OTT-MAL in vivo. Of note, Gata1 and Gata2 transcripts were also upregulated in OM versus WT cells (Supplemental Figure 4A). These results demonstrate increased RBPJ transcriptional activity in the hematopoietic progenitors of OM animals with respect to WT controls.
We next assessed whether OTT-MAL activation of RBPJ transcription is involved in the proliferation of 6133 AMKL cells. We performed coimmunoprecipitation to confirm that endogenous OTT-MAL but not MAL interacted with RBPJ in the 6133 AMKL cell line (Figure F). In addition, chromatin immunoprecipitation analyses indicated that MAL and RBPJ were present at the HES1
promoter in 6133 cells but were not present at the HES1
promoter in control Ba/F3 cells that do not express OTT-MAL (Figure G). As confirmation, we also transduced 6133 cells with a dominant negative (dn) form of RBPJ (dnRBPJ) that is unable to bind DNA (29
) or with a dn Mastermind-like 1 (dnMAML1) mutant that entraps intracellular Notch (ICN) and RBPJ in transcriptionally inactive complexes when Notch signaling is activated (30
). Compared with empty vector control, dnRBPJ expression specifically inhibited the growth of 6133 AMKL cells but not control Ba/F3 cells (Figure H and Supplemental Figure 4). In contrast, dnMAML1 or γ-secretase inhibitors had no significant effect on growth of 6133 or Ba/F3 cells (Supplemental Figure 4 and data not shown), suggesting that the expression of RBPJ target genes is not dependent on the activation of Notch receptors but is due to direct interaction between OTT-MAL and RBPJ.Taken together, these results indicate that RBPJ-mediated transcription is aberrantly induced by OTT-MAL in vitro and in vivo and is required for the growth of OTT-MAL–transformed AMKL cells.
An RBPJ pathway signature is upregulated in t(1;22)(p13;q13) AMKL.
This mouse model expressing OTT-MAL indicates that aberrant activation of the RBPJ transcription factor, a mediator of the canonical Notch signaling pathway, is important for leukemic transformation of the Mk lineage. To confirm the role of the pathway in human AMKL, we analyzed the expression of RBPJ pathway genes in human AMKL associated with t(1;22)(p13;q13) (OM-AMKL) compared with DS-AMKL, the other molecularly defined subgroup of childhood AMKL. Using published global expression data from AMKL samples (32
), we performed Gene Set Enrichment Analysis (GSEA) using a list of genes implicated in the RBPJ pathway (Supplemental Table 1). GSEA results showed that a RBPJ pathway signature was significantly enriched in OM-AMKL compared with DS-AMKL (Figure A). The most upregulated genes in OM-AMKL included Notch1
, and RBPJ
as well as direct RBPJ targets, including Hey2
, and TCFL5
) (Figure B). Together, these results show RBPJ pathway activation in human AMKL cells expressing the OTT-MAL fusion.
Gene expression analysis of human AMKL with t(1;22)(p13;q13) demonstrates a Notch pathway signature.
OTT-MAL cooperates with MPL signaling to induce AMKL in mice.
Based on the low incidence of AMKL in animals expressing OTT-MAL alone, we hypothesized that deregulation of RBPJ transcription by OTT-MAL was not sufficient for development of AMKL and that cooperating oncogenic events were required. To identify such candidate events, we first used the 6133 cell line to screen cooperating mutations in cytokine receptors and signaling molecules that have been described in human Mk malignancies (18
). This included FLT3ITDN51
, and MPLW515L
. We observed that MPLW515L
transduction but not transduction with other alleles efficiently transformed 6133 cells, resulting in cytokine-independent growth (Figure A) and induced a megakaryocytic phenotype, as assessed in part by prominent staining for AchE (Figure B).
MPL mutant transforms 6133 AMKL cells.
To compare the pathways activated by MPLW515L and by the other mutants in the context of OTT-MAL, we assessed phosphorylation levels of several relevant signaling transduction intermediates, including ERK1/2 (MAPK pathway), STAT3 and STAT5 (STAT pathway), and S6 (PI3K pathway) by flow cytometry on 6133 cells stably transduced with FLT3ITDN51, JAK2V617F, JAK2T875N, or MPLW515L, respectively. ERK1/2 was markedly more activated by MPLW515L than by the other mutants, which did not confer factor-independent growth to 6133 cells (Figure C), whereas activation of the STAT or PI3K pathway was more uniform among all the mutants (Supplemental Figure 5). To further delineate the importance of the pathway activation in this context, we tested the effect of MAPK inhibitors. We observed that the MAPK inhibitor PD98059 inhibited the growth of 6133 cells expressing MPLW515L and ERK1/2 phosphorylation in a dose-dependent manner (Figure , D and E). Of note, stimulation of 6133 cells expressing the WT MPL receptor with TPO also induced proliferation (Figure A), indicating that proliferation was mediated by activation of the TPO/MPL signaling pathway rather than specifically by the MPLW515L mutant. Interestingly, TPO stimulation of 6133 cells expressing WT MPL led to a rapid increase in Hes1 transcript but not Gata1 transcript (Figure F). This increase was inhibited by treatment with MAPK inhibitors (Figure G). Taken together, these results indicate that activation of the MPL/MAPK signaling pathway is important for megakaryoblastic transformation and increases HES1 transcription in the context of OTT-MAL.
To corroborate cooperativity between MPL signaling and OTT-MAL in primary hematopoietic cells in vivo, BM cells from 2-month-old nonleukemic OM or WT littermates were transduced with retroviruses harboring the MPLW515L
allele and transplanted into lethally irradiated WT C57BL/6 recipients (termed OM+MPLW515L
, respectively). In the C57BL/6 background BM transplant model, MPLW515L
induced an MPD with leukocytosis, polycythemia, and marked thrombocytosis similar to that reported in the BALB/c background (23
), albeit with a longer latency (median = 60 days; Supplemental Figure 6). In both the WT and OM contexts, the animals showed comparable degrees of leukocytosis, polycythemia, thrombocythemia, and splenomegaly, whereas the degree of hepatomegaly was significantly increased when
cells were used (Figure , A and B, and Supplemental Figure 6). In contrast, when MPLW515L
and OTT-MAL were coexpressed, flow cytometric analysis of splenocytes showed a significant shift toward immature progenitors as assessed by a marked increase in c-Kit+
staining in OM+MPLW515L
animals (Figure C and Supplemental Figure 7A), and the disease phenotype was more severe in this context, as indicated by a substantial increase in the number of CD41+
, and Mac1+
cells when compared with expression of MPLW515L
alone. We next performed multiparameter flow analysis on BM or spleen cells, respectively, to phenotypically characterize hematopoietic progenitor populations. In contrast with WT+MPLW515L
animals, which showed an increase in the common myeloid progenitor (CMP) population, OM+MPLW515L
animals showed a preferential increase in the Mk-erythrocyte progenitor (MEP) population in both BM and spleen (Figure D and Supplemental Figure 8). In addition, MkP that were specifically engaged in the Mk differentiation downstream of the MEP (37
) were markedly expanded in OM+MPLW515L
animals compared with WT+MPLW515L
animals (Figure E and Supplemental Figure 8).
Coexpression of MPLW515L with OTT-MAL results in AMKL in vivo.
Histopathologic analysis of BM, spleen, and liver from WT+MPLW515L or OM+MPLW515L animals showed infiltration with an admixture of megakaryocytic, erythroid, and myeloid elements (data not shown). Immunochemistry for vWF on spleen sections showed an increase in the number of mature polyploid Mks in WT+MPLW515L animals, whereas OM+MPLW515L animals showed, in addition, the presence of numerous hypolobated immature megakaryoblasts (Figure F and Supplemental Figure 9). Together, these data demonstrate that in the WT C57BL/6 background, MPLW515L induces an MPD characterized by thrombocytosis and associated with expansion of the CMP population. In contrast, coexpression of MPLW515L with OTT-MAL resulted in expansion of the MEP and MkP progenitor populations and evidence of transformation of the MPD phenotype into AMKL.
To further annotate malignant transformation, secondary transplantation was performed into sublethally irradiated recipients using 1 × 106 splenocytes from primary animals. Secondary OM+MPLW515L animals developed a disease with leukocytosis and disease latency similar to that observed in the primary OM+MPLW515L animals (Figure , A and C). However, in striking contrast with primary recipients, secondary recipients developed thrombocytopenia (Figure B). In control experiments, secondary WT+MPLW515L or OM+MPLWT animals showed no sign of disease for over 200 days, indicating that the MPLW515L-induced MPD was not transplantable. Compared with control animals, flow cytometry analysis of secondary OM+MPLW515L splenocytes showed abnormal CD41+, CD42b+, CD41/CD61+, or CD9+ populations, a fraction of which were also c-Kitlo/+ (Figure D, Supplemental Figure 7B, and Supplemental Figure 10). These cells also expressed the CD71 marker but not Ter119, Mac1, or Gr1 markers. In agreement with these observations, histopathologic analysis demonstrated that BM, spleen, and liver were heavily infiltrated with immature hypolobated vWF+ megakaryoblasts (Figure E and Supplemental Figure 10), with a striking reduction in median polyploidy (Figure F) and fibrosis (Figure G and Supplemental Figure 11). Southern blot analysis of DNA from BM cells showed over 10 viral integration sites in primary and secondary animals and suggests that the disease is oligo/polyclonal (Supplemental Figure 12). Transplantation into tertiary recipients resulted in a similar phenotype as in the secondary recipients of OM+MPLW515L cells, albeit with a shorter latency (median = 38 days; Figure C). Collectively, these data show that coexpression of OTT-MAL and an activated MPL mutant induces AMKL with characteristic features of the human disease.
OTT-MAL+MPLW515L–induced AMKL shows characteristics of human AMKL.