Cell of origin determines clinically relevant subtypes of MLL-rearranged AML.

Mixed lineage leukemia (MLL)-fusion proteins can induce acute myeloid leukemias (AMLs) from either hematopoietic stem cells (HSCs) or granulocyte-macrophage progenitors (GMPs), but it remains unclear whether the cell of origin influences the biology of the resultant leukemia. MLL-AF9-transduced single HSCs or GMPs could be continuously replated, but HSC-derived clones were more likely than GMP-derived clones to initiate AML in mice. Leukemia stem cells derived from either HSCs or GMPs had a similar immunophenotype consistent with a maturing myeloid cell (LGMP). Gene expression analyses demonstrated that LGMP inherited gene expression programs from the cell of origin including high-level Evi-1 expression in HSC-derived LGMP. The gene expression signature of LGMP derived from HSCs was enriched in poor prognosis human MLL-rearranged AML in three independent data sets. Moreover, global 5'-mC levels were elevated in HSC-derived leukemias as compared with GMP-derived leukemias. This mirrored a difference seen in 5'-mC between MLL-rearranged human leukemias that are either EVI1 positive or EVI1 negative. Finally, HSC-derived leukemias were more resistant to chemotherapy than GMP-derived leukemias. These data demonstrate that the cell of origin influences the gene expression profile, the epigenetic state and the drug response in AML, and that these differences can account for clinical heterogeneity within a molecularly defined group of leukemias.

MLL-AF9 and FLT3 cooperation in acute myelogenous leukemia: development of a model for rapid therapeutic assessment.

Human leukemias harboring chromosomal translocations involving the mixed lineage leukemia (MLL, HRX, ALL-1) gene possess high-level expression, and frequent activating mutations of the receptor tyrosine kinase FLT3. We used a murine bone marrow transplant model to assess cooperation between MLL translocation and FLT3 activation. We demonstrate that MLL-AF9 expression induces acute myelogenous leukemia (AML) in approximately 70 days, whereas the combination of MLL-AF9 and FLT3-ITD does so in less than 30 days. Secondary transplantation of splenic cells from diseased mice established that leukemia stem cells are present at a very high frequency of approximately 1:100 in both diseases. Importantly, prospectively isolated granulocyte macrophage progenitors (GMPs) coinfected with MLL-AF9 and FLT3-ITD give rise to a similar AML, with shorter latency than from GMP transduced with MLL-AF9 alone. Cooperation between MLL-AF9 and FLT3-ITD was further verified by real-time assessment of leukemogenesis using noninvasive bioluminescence imaging. We used this model to demonstrate that MLL-AF9/FLT3-ITD-induced leukemias are sensitive to FLT3 inhibition in a 2-3 week in vivo assay. These data show that activated FLT3 cooperates with MLL-AF9 to accelerate onset of an AML from whole bone marrow as well as a committed hematopoietic progenitor, and provide a new genetically defined model system that should prove useful for rapid assessment of potential therapeutics in vivo.

The ZF87/MAZ transcription factor functions as a growth suppressor in fibroblasts.

ZF87/MAZ is a zinc finger transcription factor that activates expression of tissue-specific genes and represses expression of the c-myc proto-oncogene. Infection of NIH3T3 fibroblasts with a retrovirus expressing ZF87/MAZ leads to a significant reduction in G418-resistant colonies, compared to cells infected with a retroviral control. Further, only a small fraction of the G418-resistant colonies express ZF87/MAZ. When the ZF87/MAZ-expressing colonies are expanded, they demonstrate a slow growth phenotype, a delayed transit through G1 phase and a decrease in endogenous c-myc gene expression and cyclin A and cyclin E protein levels. Consistent with a partial G1 arrest, the ZF87/MAZ-expressing cells show a reduced sensitivity to the S phase specific chemotherapeutic agent camptothecin. These data indicate that ZF87/MAZ is a growth suppressor protein in nontransformed cells, in part, by affecting the levels of key cell cycle regulatory proteins.

Transcriptional repression from the c-myc P2 promoter by the zinc finger protein ZF87/MAZ.

ZF87/MAZ is a zinc finger-containing transcription factor that was cloned based on its ability to bind to a site within the c-myc P2 promoter. However, its role in the control of c-myc transcription has not yet been well established. Here we have analyzed the effect of ZF87/MAZ overexpression on transcription from the murine c-myc P2 promoter. It was found that when overexpressed in COS cells, ZF87/MAZ significantly represses transcription from P2. The repression is mediated through the ME1a2 element, located at position -86 relative to the P2 transcriptional start site, and is not mediated through either the E2F or the ME1a1 sites. ZF87/MAZ functions as a true transcriptional repressor since it can repress transcription independently of the c-myc promoter, as part of a fusion with the GAL4 protein. The repressive domain within ZF87/MAZ is located in the amino-terminal half of the protein, a region rich in proline and alanine residues. ZF87/MAZ therefore shares features (i.e. a Pro/Ala-rich region) with those of known transcriptional repressor proteins.

An early S phase checkpoint is regulated by the E2F1 transcription factor.

The E2F1 transcription factor regulates transit of cells through the S phase checkpoint, dependent on its association with cyclin A/cdk2. Expression in cells of a mutant E2F1 lacking the cyclin A/cdk2 binding domain leads to partial arrest of cells at the S phase checkpoint. When subconfluent growing cells expressing this mutant E2F1 are analyzed in detail, it is shown here that they display a significantly reduced incorporation of 3H-thymidine into the DNA of each S phase cell, compared to control cells or to cells overexpressing full-length E2F1. Further, when cells are blocked at the G1/S phase border and released, there is a clear reduction in the amount of 3H-thymidine incorporated into the DNA of S phase cells by 1.5 hours post release. Considering a normal 6 hour S phase duration, the results show that the S phase checkpoint mediated by E2F1 is not a late S phase event but an early one.

Cyclin A levels, the duration of S phase and sensitivity to a chemotherapeutic agent are altered in fibroblasts cultured on a fibronectin matrix.

Culture of murine embryonic fibroblasts, but not vascular smooth muscle cells, on a fibronectin matrix significantly shortens their transit time through the S phase of the cell cycle. This shortening corresponds to an increase in both cyclin A protein levels and active cyclin A/cdk2 complex. The increase in cyclin A protein appears due to a translational/post-translational mechanism since there is no increase in cyclin A mRNA following culture of the cells on fibronectin. Treatment of cells cultured on fibronectin with a short pulse of the S phase chemotherapeutic agent camptothecin, resulted in a relative protection from cell death when compared to cells cultured on tissue culture plastic. Thus, while the cells have increased rate of transit through S phase fibronectin-mediated signaling protects the cells from S phase mediated apoptosis. In addition, fibroblasts constitutively expressing a mutant E2F1 transcription factor (E2F1d87) have a lengthened S phase, due to a truncation of the cyclin A/cdk2 binding domain. Culture of these mutant- expressing cells on fibronectin did not shorten their S phase duration in spite of the fact that cyclin A levels and active cyclin A/cdk2 complex were significantly elevated. Thus, although the fibronectin signaling mechanisms culminating in elevated cyclin A were intact in these mutant E2F1 expressing cells, they were insensitive to the effects of this elevated cyclin A. The effect of the mutant E2F1d87 on slowing transit through S phase appears dominant over the effect of elevated cyclin A.