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HOX genes, MEIS1, and FLT3 are frequently up-regulated in human myeloid leukemias. Meis1 cooperates with Hox genes to induce leukemias in mice, hypothetically the consequence of Meis1-induced Flt3 overexpression. To test this, we compared the properties of Flt3(-/-) and Flt3(+/+) progenitors transduced with Hoxa9 or Hoxa9/Meis1. In a myeloid clonogenic assay, Meis1 greatly enhanced the proliferation of Hoxa9-expressing cells, massively up-regulating Flt3 protein. However, the transforming potential of Hoxa9/Meis1 was unaltered in Flt3(-/-) cells. All mice that received Hoxa9/Meis1-transduced progenitors succumbed to rapid acute myeloid leukemias regardless of Flt3 genotype. Flt3 expression levels in leukemic blasts did not correlate with parameters reflecting their proliferative rate or their impaired differentiation. Furthermore, analysis of c-Myb expression levels in Hoxa9/Meis1-transformed cells showed that the up-regulation of this critical downstream effector was independent of Flt3. Altogether, our findings demonstrate that Flt3 is dispensable to the oncogenic cooperation of Meis1 with Hoxa9.

The importance of HOXA genes in T-cell acute lymphoblastic leukemia (T-ALL) has recently been recognized. We report a novel chromosomal translocation in a T-ALL patient that maps upstream of the HOXA13 gene and downstream of the BCL11B/CTIP2 locus. Analysis of HOXA gene transcription demonstrated massive expression of HOXA13, whereas the other HOXA genes were unaffected. A genomic rearrangement of the HOXA locus associated with exclusive expression of HOXA13 was observed in a second patient. This situation resembles chromosomal translocations activating genes of the TLX/HOX11 family in T-ALLs. To compare the leukemogenic properties of HOXA13 to that of TLX proteins, cohorts of lethally irradiated mice were transplanted with bone marrow transduced with a retroviral vector expressing TLX3 or HOXA13. Cells transduced with TLX3 or HOXA13 could not be detected in the peripheral blood of mice post-transplantation and none of the mice developed malignancies. Cotransduction of the HOX cofactor MEIS1 with TLX3 or HOXA13 did not alter this outcome. However, in a myeloid clonogenic assay HOXA13 and TLX3 extended the proliferation of progenitors similarly to what was observed for TLX1. Altogether, our results strongly suggest the absolute requirement for cooperative events in association with homeobox gene up-regulation to induce T-cell leukemogenesis., ((c) 2006 Wiley-Liss, Inc.)

The MLL-ELL fusion gene results from the translocation t(11;19)(q23;p13.1) that is associated with de novo and therapy-related acute myeloid leukemia. To study its transforming properties, we retrovirally transduced primary murine hematopoietic progenitors and assessed their growth properties both in vitro and in vivo. MLL-ELL increased the proliferation of myeloid colony-forming cells in methylcellulose cultures upon serial replating, whereas overexpression of ELL alone had no effect. We reconstituted lethally irradiated congenic mice with bone marrow progenitors transduced with MLL-ELL or the control MIE vector encoding the enhanced green fluorescent protein. When the peripheral blood of the mice was analyzed 11-13 weeks postreconstitution, we found that the engraftment of the MLL-ELL-transduced cells was superior to that of the MIE controls. At this time point, the contribution of the donor cells was normally distributed among the myeloid and nonmyeloid compartments. Although all of the MIE animals (n = 10) remained healthy for more than a year, all of the MLL-ELL mice (n = 20) succumbed to monoclonal or pauciclonal acute myeloid leukemias within 100-200 days. The leukemic cells were readily transplantable to secondary recipients and could be established as immortalized cell lines in liquid cultures. These studies demonstrate the enhancing effect of MLL-ELL on the proliferative potential of myeloid progenitors as well as its causal role in the genesis of acute myeloid leukemias.

Retinoic acid receptor alpha (RARalpha) is the target of several chromosomal translocations associated with acute promyelocytic leukemias (APLs). These rearrangements fuse RARalpha to different partner genes creating the chimeric proteins: PML-RARalpha, PLZF-RARalpha, and NPM-RARalpha. Although the vast majority of APLs respond to retinoic acid therapy, those associated with PLZF-RARalpha are resistant. We have used retroviruses to express PML-RARalpha, PLZF-RARalpha, NPM-RARalpha, RARalpha403 (a dominant negative mutant of RARalpha), and wild-type RARalpha in murine bone marrow progenitors and found that all of these constructs blocked differentiation and led to the immortalization of myeloid progenitors. This cellular transformation is specific to an alteration of the RARalpha pathway because overexpression of RARbeta, RARgamma, or RXRalpha did not result in similar growth perturbations. Pharmacological doses of RA induced differentiation and inhibited proliferation of cells transformed with either of the APL fusion genes, including PLZF-RARalpha, whereas physiological retinoic acid concentrations were sufficient to reverse the phenotype of cells transformed with wild-type RARalpha. The cellular responses to retinoic acid were accompanied by a sharp decrease in the amount of the RARalpha-fusion proteins expressed in the cells. Our findings suggest that the oncogenicity of RARalpha-fusion proteins results from their nature to behave as unliganded RARalpha in the presence of physiological concentrations of retinoic acid.

A subset of chromosomal translocations in acute leukemias results in the fusion of the trithorax-related protein HRX with a variety of heterologous proteins. In particular, leukemias with the t(11;19)(q23;p13.3) translocation express HRX-ENL fusion proteins and display features which suggest the malignant transformation of myeloid and/or lymphoid progenitor(s). To characterize directly the potential transforming effects of HRX-ENL on primitive hematopoietic precursors, the fusion cDNA was transduced by retroviral gene transfer into cell populations enriched in hematopoietic stem cells. The infected cells had a dramatically enhanced potential to generate myeloid colonies with primitive morphology in vitro. Primary colonies could be replated for at least three generations in vitro and established primitive myelomonocytic cell lines upon transfer into suspension cultures supplemented with interleukin-3 and stem cell factor. Immortalized cells contained structurally intact HRX-ENL proviral DNA and expressed a low-level of HRX-ENL mRNA. In contrast, wild-type ENL or a deletion mutant of HRX-ENL lacking the ENL component did not demonstrate in vitro transforming capabilities. Immortalized cells or enriched primary hematopoietic stem cells transduced with HRX-ENL induced myeloid leukemias in syngeneic and SCID recipients. These studies demonstrate a direct role for HRX-ENL in the immortalization and leukemic transformation of a myeloid progenitor and support a gain-of-function mechanism for HRX-ENL-mediated leukemogenesis.