Takeshi Inukai

Downregulation of Bim, a Proapoptotic Relative of Bcl-2, Is a Pivotal Step in Cytokine-Initiated Survival Signaling in Murine Hematopoietic Progenitors

ABSTRACT Two distinct signaling pathways regulate the survival of interleukin-3 (IL-3)-dependent hematopoietic progenitors. One originates from the membrane-proximal portion of the cytoplasmic domain of the IL-3 receptor (βc chain), which is shared by IL-3 and granulocyte-macrophage colony-stimulating factor and is involved in the regulation of Bcl-xL through activation of STAT5. The other pathway emanates from the distal region of the βc chain and overlaps with downstream signals from constitutively active Ras proteins. Although the latter pathway is indispensable for cell survival, its downstream targets remain largely undefined. Here we show that the expression of Bim, a member of the BH3-only subfamily of cell death activators, is downregulated by IL-3 signaling through either of two major Ras pathways: Raf/mitogen-activated protein kinase and the phosphatidylinositol 3-kinase/mammalian target of rapamycin. Akt/phosphokinase B does not appear to play a significant role in this regulatory cascade. Bim downregulation has important implications for cell survival, since enforced expression of this death activator at levels equivalent to those induced by cytokine withdrawal led to apoptosis even in the presence of IL-3. We conclude that Bim is a pivotal molecule in cytokine regulation of hematopoietic cell survival.

SLUG, a ces-1-Related Zinc Finger Transcription Factor Gene with Antiapoptotic Activity, Is a Downstream Target of the E2A-HLF Oncoprotein

The E2A-HLF fusion gene transforms human pro-B lymphocytes by interfering with an early step in apoptotic signaling. In a search for E2A-HLF-responsive genes, we identified a zinc finger transcription factor, SLUG, whose product belongs to the Snail family of developmental regulatory proteins. Importantly, SLUG bears close homology to the CES-1 protein of C. elegans, which acts downstream of CES-2 in a neuron-specific cell death pathway. Consistent with the postulated role of CES-1 as an antiapoptotic transcription factor, SLUG was nearly as active as Bcl-2 or Bcl-xL in promoting the survival of IL-3-dependent murine pro-B cells deprived of the cytokine. We conclude that SLUG is an evolutionarily conserved transcriptional repressor whose activation by E2A-HLF promotes the aberrant survival and eventual malignant transformation of mammalian pro-B cells otherwise slated for apoptotic death.

Slug, a highly conserved zinc finger transcriptional repressor, protects hematopoietic progenitor cells from radiation-induced apoptosis in vivo

We show here that a zinc finger transcriptional repressor, Slug, which is aberrantly upregulated by the E2A-HLF oncoprotein in pro-B cell acute leukemia, functions as an antiapoptotic factor in normal hematopoietic progenitor cells. Slug(-/-) mice were much more radiosensitive than wild-type mice, dying earlier and showing accentuated decreases in peripheral blood cell counts, as well as abundant microhemorrhages and widely disseminated bacterial microabscesses throughout the body. Slug expression was detected in diverse subsets of hematopoietic progenitors, but not in more differentiated B and T lymphoid cells, and there was a significant increase in apoptotic (TUNEL-positive) bone marrow progenitor cells in irradiated Slug(-/-) mice compared to wild-type controls. These results implicate Slug in a novel survival pathway that protects hematopoietic progenitors from apoptosis after DNA damage.

Roles of Bim in Apoptosis of Normal and Bcr-Abl-Expressing Hematopoietic Progenitors

ABSTRACT Bcr-Abl kinase is known to reverse apoptosis of cytokine-dependent cells due to cytokine deprivation, although it has been controversial whether chronic myeloid leukemia (CML) progenitors have the potential to survive under conditions in which there are limited amounts of cytokines. Here we demonstrate that early hematopoietic progenitors (Sca-1+ c-Kit+ Lin−) isolated from normal mice rapidly undergo apoptosis in the absence of cytokines. In these cells, the expression of Bim, a proapoptotic relative of Bcl-2 which plays a key role in the cytokine-mediated survival system, is induced. In contrast, those cells isolated from our previously established CML model mice resist apoptosis in cytokine-free medium without the induction of Bim expression, and these effects are reversed by the Abl-specific kinase inhibitor imatinib mesylate. In addition, the expression levels of Bim are uniformly low in cell lines established from patients in the blast crisis phase of CML, and imatinib induced Bim in these cells. Moreover, small interfering RNA that reduces the expression level of Bim effectively rescues CML cells from apoptosis caused by imatinib. These findings suggest that Bim plays an important role in the apoptosis of early hematopoietic progenitors and that Bcr-Abl supports cell survival in part through downregulation of this cell death activator.

Two Distinct Interleukin-3-Mediated Signal Pathways, Ras-NFIL3 (E4BP4) and Bcl-xL, Regulate the Survival of Murine Pro-B Lymphocytes

ABSTRACT Hematopoietic cells require cytokine-initiated signals for survival as well as proliferation. The pathways that transduce these signals, ensuring timely regulation of cell fate genes, remain largely undefined. The NFIL3 (E4BP4) transcription factor, Bcl-xL, and constitutively active mutants of components in Ras signal transduction pathways have been identified as key regulation proteins affecting murine interleukin-3 (IL-3)-dependent cell survival. Here we show that expression of NFIL3 is regulated by oncogenic Ras mutants through both the Raf–mitogen-activated protein kinase and phosphatidylinositol 3-kinase pathways. NFIL3 inhibits apoptosis without affecting Bcl-xL expression. By contrast, Bcl-xL levels are regulated through the membrane proximal portion in the cytoplasmic domain of the receptor (βc chain), which is shared by IL-3 and granulocyte-macrophage colony-stimulating factor. Activation of either pathway alone is insufficient to ensure cell survival, indicating that multiple independent signal transduction pathways mediate the survival of developing B-lymphoid cells.

p16/MTS1/INK4A gene is frequently inactivated by hypermethylation in childhood acute lymphoblastic leukemia with 11q23 translocation.

The p16 gene encoding a specific inhibitor of cyclin-dependent kinases 4 and 6 has been reported to be inactivated at a variety of rates in malignant tumors. We studied frequency and mechanism of inactivation of the p16 gene in various types of childhood acute lymphoblastic leukemia (ALL) using 36 leukemic cell lines established from children (B precursor-ALL, 28; B-ALL/Burkitt’s lymphoma, 3; and T-ALL, 5). On Southern blot, homozygous deletions or hemizygous deletions with rearrangement were detected in 14 cell lines. The expression of p16 protein was not observed on Western blot in 18 of 22 cell lines with intact p16 gene, but induced in 16 cell lines after treatment with the demethylating agent, indicating the silencing of the p16 gene by hypermethylation. Of note, the p16gene was inactivated by hypermethylation of the 5′ CpG island in nine of nine cell lines with 11q23 translocation, but was restored with the treatment of the demethylating agent. Partial methylation of the p16 gene was also demonstrated in three of eight primary leukemia samples with this translocation, suggesting that the p16 gene inactivation by hypermethylation might play a role in the leukemogenesis and disease progression of ALL with 11q23 translocation.

The JAK2 inhibitor AG490 predominantly abrogates the growth of human B-precursor leukemic cells with 11q23 translocation or Philadelphia chromosome

The Janus kinase (JAK) family is one of intracellular protein tyrosine kinases (PTKs) present in hematopoietic and lymphoid cells and has been shown to play a crucial role in a variety of biological responses. It was reported that a human B-precursor leukemic cell line was potently inhibited in its proliferation by one of synthetic PTK inhibitors (tyrphostins), AG490, via anti-JAK2 activity. However, no extensive studies about it have been performed. In the present study, we tested 16 human lymphoid leukemic cell lines (B-precursor, 12; T cell, four) for their sensitivity to AG490 usingu20023H-thymidine incorporation and colony formation assays, and found that B-precursor cell lines with 11q23 translocation or Philadelphia chromosome (Ph1) whose JAK2 proved to be constitutively phosphorylated were predominantly sensitive to AG490 at a concentration that has few inhibitory effect on normal hematopoiesis. We first revealed the association of JAK2 with BCR-ABL in Ph1-positive cell lines and with Brutons tyrosine kinase (BTK) in cell lines with 11q23 translocation by coimmunoprecipitation experiments. Of interest, AG490 markedly down-regulated phosphorylation of JAK2, but rather transiently up-regulated phosphorylation of BCR-ABL and BTK, suggesting direct implication of AG490 in the process of the JAK2 dephosphorylation. These results indicate that AG490 exerts a potent inhibitory activity to B-precursor leukemia with specific chromosomal abnormalities, and a therapeutic approach using AG490 is expected.

The E2A-HLF oncoprotein activates Groucho-related genes and suppresses Runx1.

ABSTRACT The E2A-HLF fusion gene, formed by the t(17;19)(q22;p13) chromosomal translocation in leukemic pro-B cells, encodes a chimeric transcription factor consisting of the transactivation domain of E2A linked to the bZIP DNA-binding and protein dimerization domain of hepatic leukemia factor (HLF). This oncoprotein blocks apoptosis induced by growth factor deprivation or irradiation, but the mechanism for this effect remains unclear. We therefore performed representational difference analysis (RDA) to identify downstream genetic targets of E2A-HLF, using a murine FL5.12 pro-B cell line that had been stably transfected with E2A-HLF cDNA under the control of a zinc-regulated metallothionein promoter. Two RDA clones, designated RDA1 and RDA3, were differentially upregulated in E2A-HLF-positive cells after zinc induction. The corresponding cDNAs encoded two WD40 repeat-containing proteins, Grg2 and Grg6. Both are related to the Drosophila protein Groucho, a transcriptional corepressor that lacks DNA-binding activity on its own but can act in concert with other proteins to regulate embryologic development of the fly. Expression of both Grg2 and Grg6 was upregulated 10- to 50-fold by E2A-HLF. Immunoblot analysis detected increased amounts of two additional Groucho-related proteins, Grg1 and Grg4, in cells expressing E2A-HLF. A mutant E2A-HLF protein with a disabled DNA-binding region also mediated pro-B cell survival and activated Groucho-related genes. Among the transcription factors known to interact with Groucho-related protein, only RUNX1 was appreciably downregulated by E2A-HLF. Our results identify a highly conserved family of transcriptional corepressors that are activated by E2A-HLF, and they suggest that downregulation of RUNX1 may contribute to E2A-HLF-mediated leukemogenesis.

The AD1 and AD2 Transactivation Domains of E2A Are Essential for the Antiapoptotic Activity of the Chimeric Oncoprotein E2A-HLF

ABSTRACT The chimeric oncoprotein E2A-HLF, generated by the t(17;19) chromosomal translocation in pro-B-cell acute lymphoblastic leukemia, incorporates the transactivation domains of E2A and the basic leucine zipper (bZIP) DNA-binding and protein dimerization domain of HLF (hepatic leukemic factor). The ability of E2A-HLF to prolong the survival of interleukin-3 (IL-3)-dependent murine pro-B cells after IL-3 withdrawal suggests that it disrupts signaling pathways normally responsible for cell suicide, allowing the cells to accumulate as transformed lymphoblasts. To determine the structural motifs that contribute to this antiapoptotic effect, we constructed a panel of E2A-HLF mutants and programmed their expression in IL-3-dependent murine pro-B cells (FL5.12 line), using a zinc-inducible vector. Neither the E12 nor the E47 product of the E2A gene nor the wild-type HLF protein was able to protect the cells from apoptosis induced by IL-3 deprivation. Surprisingly, different combinations of disabling mutations within the HLF bZIP domain had little effect on the antiapoptotic property of the chimeric protein, so long as the amino-terminal portion of E2A remained intact. In the context of a bZIP domain defective in DNA binding, mutants retaining either of the two transactivation domains of E2A were able to extend cell survival after growth factor deprivation. Thus, the block of apoptosis imposed by E2A-HLF in pro-B lymphocytes depends critically on the transactivating regions of E2A. Since neither DNA binding nor protein dimerization through the bZIP domain of HLF is required for this effect, we propose mechanisms whereby protein-protein interactions with the amino-terminal region of E2A allow the chimera to act as a transcriptional cofactor to alter the expression of genes regulating the apoptotic machinery in pro-B cells.

Cell transformation mediated by homodimeric E2A-HLF transcription factors.

The E2A-HLF fusion gene, created by the t(17;19)(q22;p13) chromosomal translocation in pro-B lymphocytes, encodes an oncogenic protein in which the E2A trans-activation domain is linked to the DNA-binding and protein dimerization domain of hepatic leukemia factor (HLF), a member of the proline- and acidic amino acid-rich (PAR) subfamily of bZIP transcription factors. This fusion product binds to its DNA recognition site not only as a homodimer but also as a heterodimer with HLF and two other members of the PAR bZIP subfamily, thyrotroph embryonic factor (TEF) and albumin promoter D-box binding protein (DBP). Thus, E2A-HLF could transform cells by direct regulation of downstream target genes, acting through homodimeric or heterodimeric complexes, or by sequestering normal PAR proteins into nonfunctional heterocomplexes (dominant-negative interference). To distinguish among these models, we constructed mutant E2A-HLF proteins in which the leucine zipper domain of HLF was extended by one helical turn or altered in critical charged amino acids, enabling the chimera to bind to DNA as a homodimer but not as a heterodimer with HLF or other PAR proteins. When introduced into NIH 3T3 cells in a zinc-inducible vector, each of these mutants induced anchorage-independent growth as efficiently as unaltered E2A-HLF, indicating that the chimeric oncoprotein can transform cells in its homodimeric form. Transformation also depended on an intact E2A activator region, providing further support for a gain-of-function contribution to oncogenesis rather than one based on a dominant-interfering or dominant-negative mechanism. Thus, the tumorigenic effects of E2A-HLF and its mutant forms in NIH 3T3 cells favor a straightforward model in which E2A-HLF homodimers bind directly to promoter/enhancer elements of downstream target genes and alter their patterns of expression in early B-cell progenitors.