IntroductionThe Evi-1 gene was first identified as a common locus of retroviral integration in myeloid tumors in AKXD mice. 1,2 It encodes a transcriptional regulator with 2 zinc finger domains. Evi-1 is shown to be highly expressed in human myeloid leukemias and myelodysplastic syndromes by chromosomal rearrangements involving 3q26, to which Evi-1 is mapped, 3-6 although it is expressed at a very low level in a limited stage of normal myeloid cell differentiation. The most frequent rearrangements involving 3q26 are the t(3;3)(q21; q26) and the inv(3)(q21;q26). Cases of myelodysplastic syndrome with these anomalies are characterized by the increase in the platelet count and the dysplastic features of megakaryocytes and are designated as 3q21q26 syndrome. 7 Aberrant expression of Evi-1 as a fusion transcript with AML1 (AML1/Evi-1) leads to blastic transformation in patients with chronic myelogenous leukemia. 8 Even in the absence of cytogenetically evident abnormalities at chromosome 3q26, overexpression of Evi-1 gene has been shown in a variety of myelogenous leukemias. 9 These facts strongly suggest a critical role for Evi-1 in human leukemogenesis.Our previous studies revealed that Evi-1 possesses diverse functions as an oncoprotein. [10][11][12] Among these studies is our demonstration that Evi-1 and AML1/Evi-1 repress transforming growth factor ␤ (TGF-␤) signaling. 13,14 TGF-␤ is a multifunctional peptide hormone that regulates various biological processes according to cellular contexts. In many types of cells, TGF-␤ acts as a negative regulator for cellular proliferation by inhibiting cell-cycle progression, leading to differentiation or apoptosis. In hematopoietic cells, TGF-␤ has also been shown to play an important role as a regulator of cellular growth and differentiation. Many studies show that early myeloid progenitors are sensitive to growth inhibition by TGF-␤. 15 Several cell lines derived from leukemic cells were reported to be resistant to the growth-inhibitory effect of TGF-␤, and loss of responsiveness to TGF-␤ is supposed to contribute, at least in part, to the leukemogenesis. 16 Intracellular mechanisms that transmit TGF-␤ signaling have been elucidated in detail. Upon binding of TGF-␤ to its receptors, Smad2 and Smad3, also called receptor-activated Smads (RSmads), are phosphorylated by the activated TGF-␤ receptors and oligomerize with Smad4, called common Smads (Co-Smads). Then, the complexes of R-Smads and Co-Smads accumulate in the nucleus, interact with DNA, and activate transcription of TGF-␤-responsive genes. This process is apparently simple, but many proteins, including inhibitory Smads, participate in regulating the process, and modifiy cellular responses to the stimuli. 17,18 Genetic impairments in genes encoded for the Smad proteins that strongly associate with carcinogenesis. Smad4 is deleted in about 50% of pancreatic cancers. 19 Germline mutations of human Smad4 contribute to familial juvenile polyposis, an autosomal dominant disorder characterized by predisposition to gastroi...

The Smad family proteins are critical components of the transforming growth factor (TGF)-b signaling pathway. TGF-b is a multipotent cytokine that elicits many biological functions. In particular, TGF-b exhibits e ects on the cell cycle manifested by G1-phase arrest, di erentiation, or apoptosis of several target cells, suggesting that disruption of TGF-b signaling pathway could be involved in cancer formation. Here we show one missense mutation of the Smad4 gene in the MH1 domain (P102L) and one frame shift mutation resulting in termination in the MH2 domain (D(483 ± 552)) in acute myelogeneous leukemia. Both of the mutated Smad4 proteins lack transcriptional activities. Concomitant expression of the P102L mutant with wild-type Smad4 inactivates wild-type Smad4 through inhibiting its DNA-binding ability. The D(483 ± 552) mutant blocks nuclear translocation of wild-type Smad4 and thus disrupts TGF-b signaling. This is the ®rst report showing that mutations in the Smad4 gene are associated with the pathogenesis of acute myelogeneous leukemia and the obtained results should provide useful insights into the mechanism whereby disruption of TGF-b signaling pathway could lead to acute myelogeneous leukemia. Oncogene (2001) 20, 88 ± 96.

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Acetylation of a transcription factor has recently been shown to play a significant role in gene regulation. Here we show that GATA-3 is acetylated in T cells and that a mutation introduced into amino acids 305-307 (KRR-GATA3) creates local hypoacetylation in GATA-3. Remarkably, KRR-GATA3 possesses the most potent suppressive effect when compared with other mutants that are disrupted in putative acetylation targets. Expressing this mutant in peripheral T cells results in defective T-cell homing to systemic lymphnodes, and prolonged T-cell survival after activation. These findings have significant implications in that the acetylation state of GATA-3 affects its physiological function in the immune system and, more importantly, provides evidence for the novel role of GATA-3 in T-cell survival and homing to secondary lymphoid organs.

Runx1/AML1 (also known as CBFA2 and PEBP23B) is a Runt family transcription factor critical for normal hematopoiesis. Runx1 forms a heterodimer with CBF3 and binds to the consensus PEBP2 sequence through the Runt domain. Runx1 enhances gene transcription by interacting with transcriptional coactivators such as p300 and CREB-binding protein. However, Runx1 can also suppress gene transcription by interacting with transcriptional corepressors, including mSin3A, TLE (mammalian homolog of Groucho), and histone deacetylases. Runx1 not only is critical for definitive hematopoiesis in the fetus but also is required for normal megakaryocytic maturation and T-lymphocyte and B-lymphocyte development in adult mice. Runx1 has been identified in leukemia-associated chromosomal translocations, including t(8;21) (Runx1-ETO/MTG8), t(16;21) (Runx1-MTG16), t(3;21) (Runx1-Evi1), t(12;21) (TEL-Runx1), and t(X;21) (Runx1-Fog2). The molecular mechanism of leukemogenesis by these fusion proteins is discussed. Various mutant mice expressing these fusion proteins have been created. However, expression of the fusion protein is not sufficient by itself to cause leukemia and likely requires additional events for leukemogenesis. Point mutations in a Runx1 allele cause haploinsufficiency and a biallelic null for Runx1, which are associated with familial platelet disorder with a propensity for acute myeloid leukemia (FPD/AML) and AML-M0, respectively. Thus, the correct protein structure and the precise dosage of Runx1 are essential for the maintenance of normal hematopoiesis.

The common synthetic intermediate of a potent and promising anticancer agent, fostriecin, was synthesized using a unique method that combines four catalytic asymmetric reactions as shown above.

Catalytic asymmetric synthesis of the natural antibiotic fostriecin (CI-920) and its analogue 8-epi-fostriecin and evaluation of their biological activity are described. We used four catalytic asymmetric reactions to construct all of the chiral centers of fostriecin and 8-epi-fostriecin; cyanosilylation of a ketone, Yamamoto allylation, direct aldol reaction, and Noyori reduction, two of which were developed by our group. Catalytic enantioselective cyanosilylation of ketone 13 produced the chiral tetrasubstituted carbon at C-8. Both enantiomers of the product cyanohydrin were obtained with high enantioselectivity by switching the center metal of the catalyst from titanium to gadolinium. Yamamoto allylation constructed the C-5 chiral carbon in the alpha,beta-unsaturated lactone moiety. A direct catalytic asymmetric aldol reaction of an alkynyl ketone using LLB catalyst constructed the chirality at C-9 with the introduction of a synthetically versatile alkyne moiety, which was later converted to cis-vinyl iodide, the substrate for the subsequent Stille coupling for the triene synthesis. Noyori reduction produced the secondary alcohol at C-11 from the acetylene ketone 6 with excellent selectivity. Importantly, all the stereocenters were constructed under catalyst control in this synthesis. This strategy should be useful for rapid synthesis of stereoisomers of fostriecin.

The AML1 gene encodes a DNA-binding protein that contains the runt domain and is the most frequent target of translocations associated with human leukemias. Here, point mutations of the AML1 gene, V105ter (single-letter amino acid code) and R139G, (single-letter amino acid codes) were identified in 2 cases of myelodysplastic syndrome (MDS) by means of the reverse transcriptase–polymerase chain reaction single-strand conformation polymorphism method. Both mutations are present in the region encoding the runt domain of AML1 and cause loss of the DNA-binding ability of the resultant products. Of these mutants, V105ter has also lost the ability to heterodimerize with polyomavirus enhancer binding protein 2/core binding factor β (PEBP2β/CBFβ). On the other hand, the R139G mutant acts as a dominant negative inhibitor by competing with wild-type AML1 for interaction with PEBP2β/CBFβ. This study is the first report that describes mutations of AML1 in patients with MDS and the mechanism whereby the mutant acts as a dominant negative inhibitor of wild-type AML1.