Akihide Yoshimi

Modulation of splicing catalysis for therapeutic targeting of leukemia with mutations in genes encoding spliceosomal proteins.

Mutations in genes encoding splicing factors (which we refer to as spliceosomal genes) are commonly found in patients with myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). These mutations recurrently affect specific amino acid residues, leading to perturbed normal splice site and exon recognition. Spliceosomal gene mutations are always heterozygous and rarely occur together with one another, suggesting that cells may tolerate only a partial deviation from normal splicing activity. To test this hypothesis, we engineered mice to express a mutated allele of serine/arginine-rich splicing factor 2 (Srsf2P95H)—which commonly occurs in individuals with MDS and AML—in an inducible, hemizygous manner in hematopoietic cells. These mice rapidly succumbed to fatal bone marrow failure, demonstrating that Srsf2-mutated cells depend on the wild-type Srsf2 allele for survival. In the context of leukemia, treatment with the spliceosome inhibitor E7107 (refs. 7,8) resulted in substantial reductions in leukemic burden, specifically in isogenic mouse leukemias and patient-derived xenograft AMLs carrying spliceosomal mutations. Whereas E7107 treatment of mice resulted in widespread intron retention and cassette exon skipping in leukemic cells regardless of Srsf2 genotype, the magnitude of splicing inhibition following E7107 treatment was greater in Srsf2-mutated than in Srsf2-wild-type leukemia, consistent with the differential effect of E7107 on survival. Collectively, these data provide genetic and pharmacologic evidence that leukemias with spliceosomal gene mutations are preferentially susceptible to additional splicing perturbations in vivo as compared to leukemias without such mutations. Modulation of spliceosome function may thus provide a new therapeutic avenue in genetically defined subsets of individuals with MDS or AML.

Evi1 represses PTEN expression and activates PI3K/AKT/mTOR via interactions with polycomb proteins

Evi1 (ecotropic viral integration site 1) is essential for proliferation of hematopoietic stem cells and implicated in the development of myeloid disorders. Particularly, high Evi1 expression defines one of the largest clusters in acute myeloid leukemia and is significantly associated with extremely poor prognosis. However, mechanistic basis of Evi1-mediated leukemogenesis has not been fully elucidated. Here, we show that Evi1 directly represses phosphatase and tensin homologue deleted on chromosome 10 (PTEN) transcription in the murine bone marrow, which leads to activation of AKT/mammalian target of rapamycin (mTOR) signaling. In a murine bone marrow transplantation model, Evi1 leukemia showed modestly increased sensitivity to an mTOR inhibitor rapamycin. Furthermore, we found that Evi1 binds to several polycomb group proteins and recruits polycomb repressive complexes for PTEN down-regulation, which shows a novel epigenetic mechanism of AKT/mTOR activation in leukemia. Expression analyses and ChIPassays with human samples indicate that our findings in mice models are recapitulated in human leukemic cells. Dependence of Evi1-expressing leukemic cells on AKT/mTOR signaling provides the first example of targeted therapeutic modalities that suppress the leukemogenic activity of Evi1. The PTEN/AKT/mTOR signaling pathway and the Evi1-polycomb interaction can be promising therapeutic targets for leukemia with activated Evi1.

Evi1 is essential for hematopoietic stem cell self-renewal, and its expression marks hematopoietic cells with long-term multilineage repopulating activity.

A new mouse in which an IRES-GFP cassette is knocked-in to the Evi1 locus reveals that HSC long-term multilineage repopulating activity specifically segregates with expression of the Evi1 transcription factor.

Positive feedback between NF-κB and TNF-α promotes leukemia-initiating cell capacity

Acute myeloid leukemia (AML) is a heterogeneous hematologic malignancy that originates from leukemia-initiating cells (LICs). The identification of common mechanisms underlying LIC development will be important in establishing broadly effective therapeutics for AML. Constitutive NF-κB pathway activation has been reported in different types of AML; however, the mechanism of NF-κB activation and its importance in leukemia progression are poorly understood. Here, we analyzed myeloid leukemia mouse models to assess NF-κB activity in AML LICs. We found that LICs, but not normal hematopoietic stem cells or non-LIC fractions within leukemia cells, exhibited constitutive NF-κB activity. This activity was maintained through autocrine TNF-α secretion, which formed an NF-κB/TNF-α positive feedback loop. LICs had increased levels of active proteasome machinery, which promoted the degradation of IκBα and further supported NF-κB activity. Pharmacological inhibition of the proteasome complex markedly suppressed leukemia progression in vivo. Conversely, enhanced activation of NF-κB signaling expanded LIC frequency within leukemia cell populations. We also demonstrated a strong correlation between NF-κB activity and TNF-α secretion in human AML samples. Our findings indicate that NF-κB/TNF-α signaling in LICs contributes to leukemia progression and provide a widely applicable approach for targeting LICs.

Evi-1 is a transcriptional target of mixed-lineage leukemia oncoproteins in hematopoietic stem cells

Ecotropic viral integration site-1 (Evi-1) is a nuclear transcription factor that plays an essential role in the regulation of hematopoietic stem cells. Aberrant expression of Evi-1 has been reported in up to 10% of patients with acute myeloid leukemia and is a diagnostic marker that predicts a poor outcome. Although chromosomal rearrangement involving the Evi-1 gene is one of the major causes of Evi-1 activation, overexpression of Evi-1 is detected in a subgroup of acute myeloid leukemia patients without any chromosomal abnormalities, which indicates the presence of other mechanisms for Evi-1 activation. In this study, we found that Evi-1 is frequently up-regulated in bone marrow cells transformed by the mixed-lineage leukemia (MLL) chimeric genes MLL-ENL or MLL-AF9. Analysis of the Evi-1 gene promoter region revealed that MLL-ENL activates transcription of Evi-1. MLL-ENL-mediated up-regulation of Evi-1 occurs exclusively in the undifferentiated hematopoietic population, in which Evi-1 particularly contributes to the propagation of MLL-ENL-immortalized cells. Furthermore, gene-expression analysis of human acute myeloid leukemia cases demonstrated the stem cell-like gene-expression signature of MLL-rearranged leukemia with high levels of Evi-1. Our findings indicate that Evi-1 is one of the targets of MLL oncoproteins and is selectively activated in hematopoietic stem cell-derived MLL leukemic cells.

A role for RUNX1 in hematopoiesis and myeloid leukemia.

Since its discovery from a translocation in leukemias, the runt-related transcription factor 1/acute myelogenous leukemia-1 (RUNX1/AML1), which is widely expressed in hematopoietic cells, has been extensively studied. Many lines of evidence have shown that RUNX1 plays a critical role in regulating the development and precise maintenance of mammalian hematopoiesis. Studies using knockout mice have shown the importance of RUNX1 in a wide variety of hematopoietic cells, including hematopoietic stem cells and megakaryocytes. Recently, target molecular processes of RUNX1 in normal and malignant hematopoiesis have been revealed. Although RUNX1 is not required for the maintenance of hematopoietic stem cells, it is required for the homeostasis of hematopoietic stem and progenitor cells, and expansion of hematopoietic stem and progenitor cells due to RUNX1 deletion may be an important cause of human leukemias. Molecular abnormalities cooperating with loss of RUNX1 have also been identified. These findings may lead to a further understanding of human leukemias, and suggest novel molecular targeted therapies in the near future.

Pbx1 is a downstream target of Evi-1 in hematopoietic stem/progenitors and leukemic cells.

Ecotropic viral integration site-1 (Evi-1) is a nuclear transcription factor, which is essential for the proliferation/maintenance of hematopoietic stem cells (HSCs). Aberrant expression of Evi-1 has been frequently found in myeloid leukemia, and is associated with a poor patient survival. Recently, we reported candidate target genes of Evi-1 shared in HSCs and leukemic cells using gene expression profiling analysis. In this study, we identified Pbx1, a proto-oncogene in hematopoietic malignancy, as a target gene of Evi-1. Overexpression of Evi-1 increased Pbx1 expression in hematopoietic stem/progenitor cells. An analysis of the Pbx1 promoter region revealed that Evi-1 upregulates Pbx1 transcription. Furthermore, reduction of Pbx1 levels through RNAi-mediated knockdown significantly inhibited Evi-1-induced transformation. In contrast, knockdown of Pbx1 did not impair bone marrow transformation by E2A/HLF or AML1/ETO, suggesting that Pbx1 is specifically required for the maintenance of bone marrow transformation mediated by Evi-1. These results indicate that Pbx1 is a target gene of Evi-1 involved in Evi-1-mediated leukemogenesis.

Recurrent CDC25C mutations drive malignant transformation in FPD/AML

Familial platelet disorder (FPD) with predisposition to acute myelogenous leukaemia (AML) is characterized by platelet defects with a propensity for the development of haematological malignancies. Its molecular pathogenesis is poorly understood, except for the role of germline RUNX1 mutations. Here we show that CDC25C mutations are frequently found in FPD/AML patients (53%). Mutated CDC25C disrupts the G2/M checkpoint and promotes cell cycle progression even in the presence of DNA damage, suggesting a critical role for CDC25C in malignant transformation in FPD/AML. The predicted hierarchical architecture shows that CDC25C mutations define a founding pre-leukaemic clone, followed by stepwise acquisition of subclonal mutations that contribute to leukaemia progression. In three of seven individuals with CDC25C mutations, GATA2 is the target of subsequent mutation. Thus, CDC25C is a novel gene target identified in haematological malignancies. CDC25C is also useful as a clinical biomarker that predicts progression of FPD/AML in the early stage.

Key roles of histone methyltransferase and demethylase in leukemogenesis

A growing body of evidence has underlined the involvement of histone methyltransferases and demethylases in leukemia development. These findings can be roughly classified into two categories according to their association with leukemia. On the one hand, these histone modifiers are recruited to DNA by specific affinities of aberrantly expressed transcription factors or fusion proteins, and induce chromatin modifications to regulate target gene expression. Epigenetic regulators may function as oncogenes in this context. On the other hand, recent studies have identified inactivating mutations of some key histone modulators in myeloid malignancies and these results suggest that they act as tumor suppressors. Profound understanding of these findings in the two categories will help us consider clinical applications of epigenetic drugs. In this prospect we will review the leukemogenic mechanisms clarified by the epigenetic approach and the current findings on genetic aberrations in each methyltransferase or demethylase, and discuss the potential of medical intervention in leukemia or leukemia stem cells targeting histone modifiers. J. Cell. Biochem. 112: 415–424, 2011.

EVI1 oncogene promotes KRAS pathway through suppression of microRNA-96 in pancreatic carcinogenesis

Despite frequent KRAS mutation, the early molecular mechanisms of pancreatic ductal adenocarcinoma (PDAC) development have not been fully elucidated. By tracking a potential regulator of another feature of PDAC precursors, acquisition of foregut or gastric epithelial gene signature, we herein report that aberrant overexpression of ecotropic viral integration site 1 (EVI1) oncoprotein, which is usually absent in normal pancreatic duct, is a widespread marker across the full spectrum of human PDAC precursors and PDAC. In pancreatic cancer cells, EVI1 depletion caused remarkable inhibition of cell growth and migration, indicating its oncogenic roles. Importantly, we found that EVI1 upregulated KRAS expression through suppression of a potent KRAS suppressor, miR-96, in pancreatic cancer cells. Collectively, the present findings suggest that EVI1 overexpression and KRAS mutation converge on activation of the KRAS pathway in early phases of pancreatic carcinogenesis and propose EVI1 and/or miR-96 as early markers and therapeutic targets in this dismal disease.