Runx1 is essential at two stages of early murine B-cell development.

The t(12;21) chromosomal translocation, targeting the gene encoding the RUNX1 transcription factor, is observed in 25% of pediatric acute lymphoblastic leukemia (ALL) and is an initiating event in the disease. To elucidate the mechanism by which RUNX1 disruption initiates leukemogenesis, we investigated its normal role in murine B-cell development. This study revealed 2 critical functions of Runx1: (1) to promote survival and development of progenitors specified to the B-cell lineage, a function that can be substituted by ectopic Bcl2 expression, and (2) to enable the developmental transition through the pre-B stage triggered by the pre-B-cell antigen receptor (pre-BCR). Gene expression analysis and genomewide Runx1 occupancy studies support the hypothesis that Runx1 reinforces the transcription factor network governing early B-cell survival and development and specifically regulates genes encoding members of the Lyn kinase subfamily (key integrators of interleukin-7 and pre-BCR signaling) and the stage-specific transcription factors SpiB and Aiolos (critical downstream effectors of pre-BCR signaling). Interrogation of expression databases of 257 ALL samples demonstrated the specific down-regulation of the SPIB and IKZF3 genes (the latter encoding AIOLOS) in t(12;21) ALL, providing novel insight into the mechanism by which the translocation blocks B-cell development and promotes leukemia.

CD133 marks a stem cell population that drives human primary myelofibrosis.

Primary myelofibrosis is a myeloproliferative neoplasm characterized by bone marrow fibrosis, megakaryocyte atypia, extramedullary hematopoiesis, and transformation to acute myeloid leukemia. To date the stem cell that undergoes the spatial and temporal chain of events during the development of this disease has not been identified. Here we describe a CD133(+) stem cell population that drives the pathogenesis of primary myelofibrosis. Patient-derived circulating CD133(+) but not CD34(+)CD133(-) cells, with a variable burden for JAK2 (V617F) mutation, had multipotent cloning capacity in vitro. CD133(+) cells engrafted for up to 10 months in immunocompromised mice and differentiated into JAK2-V617F(+) myeloid but not lymphoid progenitors. We observed the persistence of human, atypical JAK2-V617F(+) megakaryocytes, the initiation of a prefibrotic state, bone marrow/splenic fibrosis and transition to acute myeloid leukemia. Leukemic cells arose from a subset of CD133(+) cells harboring EZH2 (D265H) but lacking a secondary JAK2 (V617F) mutation, consistent with the hypothesis that deregulation of EZH2 activity drives clonal growth and increases the risk of acute myeloid leukemia. This is the first characterization of a patient-derived stem cell population that drives disease resembling both chronic and acute phases of primary myelofibrosis in mice. These results reveal the importance of the CD133 antigen in deciphering the neoplastic clone in primary myelofibrosis and indicate a new therapeutic target for myeloproliferative neoplasms.

Cloning of genes involved in chromosomal translocations by high-resolution single nucleotide polymorphism genomic microarray.

High-resolution single nucleotide polymorphism genomic microarray (SNP-chip) is a useful tool to define gene dosage levels over the whole genome, allowing precise detection of deletions and duplications/amplifications of chromosomes in cancer cells. We found that this new technology can also identify breakpoints of chromosomes involved in unbalanced translocations, leading to identification of fusion genes. Using this technique, we found that the PAX5 gene was rearranged to a variety of partner genes including ETV6, FOXP1, AUTS2, and C20orf112 in pediatric acute lymphoblastic leukemia (ALL). The 3' end of the PAX5 gene was replaced by the partner gene. The PAX5 fusion products bound to PAX5 recognition sequences as strongly as wild-type PAX5 and suppressed its transcriptional activity in a dominant-negative fashion. In human B cell leukemia cells, binding of wild-type PAX5 to a regulatory region of BLK, one of the direct downstream target genes of PAX5, was diminished by expression of the PAX5-fusion protein, leading to repression of BLK. Expression of PAX5-fusion genes in murine bone marrow cells blocked development of mature B cells. PAX5-fusion proteins may contribute to leukemogenesis by blocking differentiation of hematopoietic cells into mature B cells. SNP-chip is a powerful tool to identify fusion genes in human cancers.

RUNX1 DNA-binding mutants, associated with minimally differentiated acute myelogenous leukemia, disrupt myeloid differentiation.

Mutations in the RUNX1 gene are found at high frequencies in minimally differentiated acute myelogenous leukemia. In addition to null mutations, many of the mutations generate Runx1 DNA-binding (RDB) mutants. To determine if these mutants antagonize wild-type protein activity, cDNAs were transduced into murine bone marrow or human cord blood cells using retroviral vectors. Significantly, the RDB mutants did not act in a transdominant fashion in vivo to disrupt Runx1 activity in either T-cell or platelet development, which are highly sensitive to Runx1 dosage. However, RDB mutant expression impaired expansion and differentiation of the erythroid compartment in which Runx1 expression is normally down-regulated, showing that a RDB-independent function is incompatible with erythroid differentiation. Significantly, both bone marrow progenitors expressing RDB mutants or deficient for Runx1 showed increased replating efficiencies in vitro, accompanied by the accumulation of myeloblasts and dysplastic progenitors, but the effect was more pronounced in RDB cultures. Disruption of the interface that binds CBFbeta, an important cofactor of Runx1, did not impair RDB mutant replating activity, arguing against inactivation of Runx1 function by CBFbeta sequestration. We propose that RDB mutants antagonize Runx1 function in early progenitors by disrupting a critical balance between DNA-binding-independent and DNA-binding-dependent signaling.

Defining the oncogenic function of the TEL/AML1 (ETV6/RUNX1) fusion protein in a mouse model.

The t(12;21) translocation, generating the TEL/AML1 fusion protein, is the most common genetic lesion in childhood cancer. Using a bone marrow transplantation model, we demonstrate that TEL/AML1 expression impinges on normal hematopoietic differentiation, leading to the in vivo accumulation and persistence of an early progenitor compartment with a Sca1(+)/Kit(hi)/CD11b(+) phenotype and an increased self-renewal capacity, as documented by replating assays in vitro. Differentiation of these cells is not blocked, but the frequency of mature blood cells arising from TEL/AML1-transduced progenitors is low. Impaired differentiation is prominently observed in the pro-B-cell compartment, resulting in an proportional increase in early progenitors in vivo, consistent with the t(12;21) ALL phenotype. Despite the accumulation of both multipotent and B-cell progenitors in vivo, no leukemia induction was observed during an observation period of over 1 year. These results are consistent with findings in twins with concordant ALL, showing that TEL/AML1 generates a preleukemic clone in utero that persists for several years in a clinically covert fashion. Furthermore, our studies showed that the pointed domain of TEL/AML1, which recruits transcriptional repressors and directs oligomerization with either TEL/AML1 or wild-type TEL, was essential for the observed differentiation impairment and could not be replaced with another oligomerization domain.

RGB marking facilitates multicolor clonal cell tracking.

We simultaneously transduced cells with three lentiviral gene ontology (LeGO) vectors encoding red, green or blue fluorescent proteins. Individual cells were thereby marked by different combinations of inserted vectors, resulting in the generation of numerous mixed colors, a principle we named red-green-blue (RGB) marking. We show that lentiviral vector-mediated RGB marking remained stable after cell division, thus facilitating the analysis of clonal cell fates in vitro and in vivo. Particularly, we provide evidence that RGB marking allows assessment of clonality after regeneration of injured livers by transplanted primary hepatocytes. We also used RGB vectors to mark hematopoietic stem/progenitor cells that generated colored spleen colonies. Finally, based on limiting-dilution and serial transplantation assays with tumor cells, we found that clonal tumor cells retained their specific color-code over extensive periods of time. We conclude that RGB marking represents a useful tool for cell clonality studies in tissue regeneration and pathology.

Adaptor protein Lnk inhibits c-Fms-mediated macrophage function.

The M-CSFR (c-Fms) participates in proliferation, differentiation, and survival of macrophages and is involved in the regulation of distinct macrophage functions. Interaction with the ligand M-CSF results in phosphorylation of tyrosine residues on c-Fms, thereby creating binding sites for molecules containing SH2 domains. Lnk is a SH2 domain adaptor protein that negatively regulates hematopoietic cytokine receptors. Here, we show that Lnk binds to c-Fms. Biological and functional effects of this interaction were examined in macrophages from Lnk-deficient (KO) and WT mice. Clonogenic assays demonstrated an elevated number of M-CFUs in the bone marrow of Lnk KO mice. Furthermore, the M-CSF-induced phosphorylation of Akt in Lnk KO macrophages was increased and prolonged, whereas phosphorylation of Erk was diminished. Zymosan-stimulated production of ROS was increased dramatically in a M-CSF-dependent manner in Lnk KO macrophages. Lastly, Lnk inhibited M-CSF-induced migration of macrophages. In summary, we show that Lnk binds to c-Fms and can blunt M-CSF stimulation. Modulation of levels of Lnk in macrophages may provide a unique therapeutic approach to increase innate host defenses.

Gatekeeper function of the RUNX1 transcription factor in acute leukemia.

The RUNX1 gene encodes the alpha subunit of the core binding factor (CBF) and is a common target of genetic mutations in acute leukemia. We propose that RUNX1 is a gatekeeper gene, the disruption of which leads to the exodus of a subset of hematopoietic progenitors with increased self-renewal potential from the normal environmental controls of homeostasis. This pool of "escaped" cells is the target of secondary mutations, accumulating over time to induce the aggressive manifestation of acute leukemia. Evidence from patient and animal studies supports the concept that RUNX1 mutations are the initiating event in different leukemia subtypes, but also suggests that diverse mechanisms are used to subvert RUNX1 function. One common result is the inhibition of differentiation-but its effect impinges on different lineages and stages of differentiation, depending on the mutation or fusion partner. A number of different approaches have led to the identification of secondary events that lead to the overt acute phase; however, the majority is unknown. Finally, the concept of the "leukemia stem cell" and its therapeutic importance is discussed in light of the RUNX1 gatekeeper function.