Specific chromosome translocations are frequently found in human leukemias and often result in the expression of fusion gene products. Research at the Molecular Oncology Division focuses on the molecular mechanisms of leukemia pathogenesis through functional analyses of leukemia-associated factors which could become molecular targets for leukemia treatment.

Acute promyelocytic leukemia (APL) has been characterized as a catastrophic termination of differentiation at the promyelocyte stage associated with the t(15;17) chromosomal translocation that generates a promyelocytic leukemia (PML)- retinoic acid receptor-a (RARA) fusion protein. PML is a nuclear protein that functions as a regulator of transcription, cell proliferation, apoptosis, and myeloid cell differentiation. To clarify the role of PML-RARA in the pathogenesis of APL, especially to clarify how it causes the arrest of differentiation, the biological properties of PML-RARA and its effects on normal PML function were investigated. PML-deficient mice have an impaired capacity for terminal maturation of their myeloid precursor cells. This finding has been explained to be due, at least in part, to the lack of PML action to modulate the retinoic acid-mediated differentiating activities. C/EBPepsilon expression is reduced in PML-deficient mice. PU.1 directly activates the transcription of the C/EBPepsilon gene that is essential for granulocytic differentiation. The type IV isoform of PML interacts with PU.1, promotes its association with p300, and then enhances PU.1-induced transcription and granulocytic differentiation. On the other hand, PML-RARA dissociates the PU.1/PML IV/p300 complex and inhibits PU.1-induced transcription, suggesting that PML-RARA acts as a dominant-negative inhibitor of PML-induced transcription. These results suggest a novel pathogenic mechanism of the PML-retinoic acid receptor alpha fusion protein in acute promyelocytic leukemia ⁽⁶³⁾.

PML is subjected to posttranslational modifications such as sumoylation and phosphorylation. However, the physiological significance of these modifications, especially in myeloid cell differentiation, remains unclear. In one study, we found high levels of phosphorylation of four serine residues in the PML C-terminal region in a myeloid cell line. Wild-type PML accelerated G-CSF-induced granulocytic differentiation, whereas a phosphorylation- deficient PML mutant failed to exert this action. PML interacted with C/EBPe, a transcription factor essential for granulopoiesis, activated C/EBPe-mediated transcription in concert with p300, and accelerated C/EBPe-induced granulocytic differentiation. Phosphorylation of PML was required for stimulating C/EBPe- dependent transcription and accelerating C/EBPe- induced granulocytic differentiation. We also found that PML phosphorylation was required for stimulation of PU.1-dependent transcription and acceleration of PU.1-induced granulocytic differentiation. These results suggest that phosphorylation plays essential roles in the regulation of PML to accelerate granulocytic differentiation through multiple pathways.

The AML1 transcription factor complex is the most frequent target of leukemia-associated chromosomal translocations. Homeodomain- interacting protein kinase 2 (HIPK2) is a part of the AML1 complex and activates AML1-mediated transcription. However, chromosomal translocations and mutations of HIPK2 have not been reported. In the current study, we screened mutations of the HIPK2 gene in 50 cases of acute myeloid leukemia (AML) and 80 cases of myelodysplastic syndrome (MDS). The results indicated that there were two missense mutations (R868W and N958I) in the speckle-retention signal (SRS) domain of HIPK2, which is required for localization of HIPK2 in the nuclear speckles. Subcellular localization analyses indicated that the two mutants were largely localized to nuclear regions in a conical or ring shape, and were somewhat diffused in the nucleus, in contrast to the finding in the wild type, in which were mainly localized in the nuclear speckles. The mutations interfered with the overlapping localization of AML1 and HIPK2. The mutants also showed decreased activities and inhibites wild-type HIPK2-mediated activation in AML1- and p53-dependent transcription. These findings suggest that dysfunction of HIPK2 may play a role in the pathogenesis of leukemia ⁽⁶⁴⁾.

The HIPK2 gene has been mapped to the human chromosome 7q32-q34. Deletion of 7q is frequently found in AML and MDS. Initially, we expected mutation of HIPK2 in patients with the 7q deletion, with resultant homozygous loss of functional HIPK2. However, no mutations were found in these types of patients. As a matter of fact, patients with HIPK2 mutations showed normal karyotypes and did not have any other mutations in AML1. These results suggest that the mutation of HIPK2 plays a role similar to chromosome translocations and AML1 mutations in the pathogenesis of leukemia. HIPK2 interactions result in the phosphorylation of AML1 and p300 leading to stimulation of AML1-mediated transcription. The two missense mutants that we identified here (R868W and N958I) showed decreased activities in AML1- and p53-dependent transcription. However, the R868W and N958I mutants affected neither the kinase activity nor the interaction with AML1 and p300. The R868W and N958I mutations were found in the SRS domain, which is reportedly associated with the HIPK2 localization in the nuclear speckles. In fact, R868W and N958I mutations showed impaired localization of HIPK2 in the nuclear speckles. Deletion analysis of HIPK2 indicated that the SRS domain is required for HIPK2 to stimulate AML1-mediated transcription activation. These data suggest that the mutations destabilize the localization of HIPK2, leading to dysfunction of the AML1 transcription factor complex ⁽⁶⁵⁾.

Naturally arising CD25+CD4+ regulatory T cells (T(R) cells) are involved in the maintenance of immunological self-tolerance and immune homeostasis by suppressing aberrant or excessive immune responses, such as autoimmune disease and allergy. T(R) cells specifically express the transcription factor Foxp3, a key regulator of T(R)-cell development and function. Ectopic expression of Foxp3 in conventional T cells was found to be sufficient to confer suppressive activity, repress the production of cytokines such as interleukin-2 (IL-2) and interferon-gamma (IFN-gamma), and upregulate T(R)-cell- associated molecules such as CD25, cytotoxic T-lymphocyte-associated antigen-4, and glucocorticoid-induced TNF-receptor-family- related protein. However, the method by which Foxp3 controls these molecular events has yet to be clarified. Here, we show that AML1 activates IL-2 and IFN-gamma gene expression in conventional CD4+ T cells through binding to their respective promoters. In natural T(R) cells, Foxp3 interacts physically with AML1. Several lines of evidence support a model in which the interaction suppresses IL-2 and IFN-gamma production, upregulates T(R)-cell- associated molecules, and exerts suppressive activity. This transcriptional control of T(R)-cell function by an interaction between Foxp3 and AML1 can be exploited to control physiological and pathological T-cell-mediated immune responses.