Ai Kotani

Role of AID in tumorigenesis.

A hallmark of mature B-cell lymphomas is reciprocal chromosomal translocations involving the Ig locus and a proto-oncogene, which usually result in the deregulated, constitutive expression of the translocated gene. In addition to such translocations, proto-oncogenes are frequently hypermutated in germinal center (GC)-derived B-cell lymphomas. Although aberrant, mistargeted class switch recombination (CSR) and somatic hypermutation (SHM) events have long been suspected of causing chromosomal translocations and mutations in oncogenes, and thus of playing a critical role in the pathogenesis of most B-cell lymphomas, the molecular basis for such deregulation of CSR and SHM is only beginning to be elucidated by recent genetic approaches. The tumorigenic ability of activation-induced cytidine deaminase (AID), a key enzyme that initiates CSR and SHM, was revealed in studies on AID transgenic mice. In addition, experiments with AID-deficient mice clearly showed that AID is required not only for the c-myc/IgH translocation but also for the malignant progression of translocation-bearing lymphoma precursor cells, probably by introducing additional genetic hits. Normally, AID expression is only transiently and specifically induced in activated B cells in GCs. However, recent studies indicate that AID can be induced directly in B cells outside the GCs by various pathogens, including transforming viruses associated with human malignancies. Indeed, AID expression is not restricted to GC-derived B-cell lymphomas, but is also found in other types of B-cell lymphoma and even in nonlymphoid tumors, suggesting that ectopically expressed AID is involved in tumorigenesis and disease progression in a wide variety of cell types.

miR-128b is a potent glucocorticoid sensitizer in MLL-AF4 acute lymphocytic leukemia cells and exerts cooperative effects with miR-221

MLL-AF4 acute lymphocytic leukemia (ALL) has a poor prognosis. MicroRNAs (miRNA) are small noncoding RNAs that posttranscriptionally regulate expression of target mRNAs. Our analysis of previously published data showed that expression of miR-128b and miR-221 is down-regulated in MLL-rearranged ALL relative to other types of ALL. Reexpression of these miRNAs cooperatively sensitizes 2 cultured lines of MLL-AF4 ALL cells to glucocorticoids. Target genes down-regulated by miR-128b include MLL, AF4, and both MLL-AF4 and AF4-MLL fusion genes; miR-221 down-regulates CDKN1B. These results demonstrate that down-regulation of miR-128b and miR-221 is implicated in glucocorticoid resistance and that restoration of their levels is a potentially promising therapeutic in MLL-AF4 ALL.

Activation-induced cytidine deaminase (AID) promotes B cell lymphomagenesis in Emu-cmyc transgenic mice

Activation-induced cytidine deaminase (AID), which is essential to both class switch recombination and somatic hypermutation of the Ig gene, is expressed in many types of human B cell lymphoma/leukemia. AID is a potent mutator because it is involved in DNA breakage not only of Ig but also of other genes, including proto-oncogenes. Recent studies suggest that AID is required for chromosomal translocation involving cmyc and Ig loci. However, it is unclear whether AID plays other roles in tumorigenesis. We examined the effect of AID deficiency on the generation of surface Ig-positive B cell lymphomas in Emu-cmyc transgenic mice. Almost all lymphomas that developed in AID-deficient transgenic mice were pre-B cell lymphomas, whereas control transgenic mice had predominantly B cell lymphomas, indicating that AID is required for development of B but not pre-B cell lymphomas from cmyc overexpressing tumor progenitors. Thus, AID may play multiple roles in B cell lymphomagenesis.

A novel mutation in the miR-128b gene reduces miRNA processing and leads to glucocorticoid resistance of MLL-AF4 acute lymphocytic leukemia cells

MLL-AF4 Acute Lymphocytic Leukemia has a poor prognosis, and the mechanisms by which these leukemias develop are not understood despite intensive research based on well-known concepts and methods. MicroRNAs (miRNAs) are a new class of small noncoding RNAs that post-transcriptionally regulate expression of target mRNA transcripts. We recently reported that ectopic expression of miR-128b together with miR-221, two of the miRNAs downregulated in MLL-AF4 ALL, restores glucocorticoid resistance through downregulation of the MLL-AF4 chimeric fusion proteins MLL-AF4 and AF4-MLL that are generated by chromosomal translocation t(4;11). Here we report the identification of new mutations in miR-128b in RS4;11 cells, derived from MLL-AF4 ALL patient. One novel mutation significantly reduces the processing of miR-128b. Finally, this base change occurs in a primary MLL-AF4 ALL sample as an acquired mutation. These results demonstrate that the novel mutation in miR-128b in MLL-AF4 ALL alters the processing of miR-128b and that the resultant downregulation of mature miR-128b contributes to glucocorticoid resistance through the failure to downregulate the fusion oncogenes.

Signaling of gp34 (OX40 ligand) induces vascular endothelial cells to produce a CC chemokine RANTES/CCL5.

We previously showed that gp34 (OX40 ligand) expressed on vascular endothelial cells is not only involved in adhesion between activated T cells and endothelial cells but also by itself able to transmit intracellular signals leading to expression of c-fos and c-jun mRNA upon OX40 binding. In the present study, we searched for genes that were induced or upregulated by gp34 signaling in human umbilical vein endothelial cells (HUVECs) to define its downstream biological events. HUVECs expressing high levels of gp34 were stimulated with recombinant soluble OX40 or mock control and subjected to analysis using cDNA expression arrays. We found that a CC chemokine RANTES (regulated upon activation, normal T cell expressed and secreted)/CCL5 is one of such inducible genes. Reverse transcriptase-PCR analysis showed that expression of RANTES mRNA was induced after incubation with soluble OX40 and this induction was inhibited by anti-gp34 mAb. We could detect expression of intracellular RANTES protein by flow cytometry in HUVECs stimulated with soluble OX40 as well as fixed OX40 transfectant cells but not those stimulated with mock supernatants or mock transfectant cells. Again, this induction of RANTES protein was inhibited by anti-gp34 mAb. These results clearly indicate that gp34 signaling induces expression of RANTES at both mRNA and protein levels in HUVECs and suggest a possible link between the OX40/gp34 system and RANTES during the process of T cell adhesion to endothelial cells and subsequent extravasation.

Alteration of processing induced by a single nucleotide polymorphism in pri-miR-126.

MicroRNAs (miRNAs) are small non-coding RNAs that inhibit expression of specific target genes at the post-transcriptional level. Sequence variations in miRNA genes, including pri-miRNAs, pre-miRNAs and mature miRNAs, could potentially influence the processing and/or target selection of miRNAs. In this study, we have found the single nucleotide polymorphism (SNP) at the twenty-fourth nucleotide (+24) of the mature miR-126 in the genome of RS4;11 cells, derived from a MLL-AF4 ALL patient. Through a series of in vivo analyzes, we found that this miR-126 SNP significantly blocks the processing of pri-miRNA to mature miRNA, as well as reduces miRNA-mediated translational suppression. Moreover, its frequency is different among races. Thus, our study emphasizes the importance of identifying new miRNA SNP and its contribution to miRNA biogenesis which is possible link to human genetic disease.

The AID dilemma: infection, or cancer?

Activation-induced cytidine deaminase (AID), which is both essential and sufficient for forming antibody memory, is also linked to tumorigenesis. AID is found in many B lymphomas, in myeloid leukemia, and in pathogen-induced tumors such as adult T cell leukemia. Although there is no solid evidence that AID causes human tumors, AID-transgenic and AID-deficient mouse models indicate that AID is both sufficient and required for tumorigenesis. Recently, AIDs ability to cleave DNA has been shown to depend on topoisomerase 1 (Top1) and a histone H3K4 epigenetic mark. When the level of Top1 protein is decreased by AID activation, it induces irreversible cleavage in highly transcribed targets. This finding and others led to the idea that there is an evolutionary link between meiotic recombination and class switch recombination, which share H3K4 trimethyl, topoisomerase, the MRN complex, mismatch repair family proteins, and exonuclease 3. As Top1 has recently been shown to be involved in many transcription-associated genome instabilities, it is likely that AID took advantage of basic genome instability or diversification to evolve its mechanism for immune diversity. AID targets are therefore not highly specific to immunoglobulin genes and are relatively abundant, although they have strict requirements for transcription-induced H3K4 trimethyl modification and repetitive sequences prone to forming non-B structures. Inevitably, AID-dependent cleavage takes place in nonimmunoglobulin targets and eventually causes tumors. However, battles against infection are waged in the context of acute emergencies, while tumorigenesis is rather a chronic, long-term process. In the interest of survival, vertebrates must have evolved AID to prevent infection despite its long-term risk of causing tumorigenesis.

miRNAs in normal and malignant B cells

MicroRNAs (miRNA) are small noncoding RNAs that posttranscriptionally regulate expression of target mRNAs. In animals, miRNAs control many developmental and physiological processes. It has recently revealed that miRNAs critically work in B cell development and some B cell malignancy and that their expression profiles well correlate with its prognosis and phenotype. This review overviews these interesting findings and discuss a possibility that miRNA is a promising target for therapeutic and diagnostic tool for B cell malignancy.

Novel functional small RNAs are selectively loaded onto mammalian Ago1

Argonaute (Ago) proteins function in RNA silencing as components of the RNA-induced silencing complex (RISC). In lower organisms, the small interfering RNA and miRNA pathways diverge due in part to sorting mechanisms that direct distinct small RNA (sRNA) duplexes onto specific Ago-RISCs. However, such sorting mechanisms appear to be lost in mammals. miRNAs appear not to distinguish among Ago1–4. To determine the effect of viral infection on the sorting system, we compared the content of deep-sequenced RNA extracted from immunoprecipitation experiments with the Ago1 and Ago2 proteins using Epstein–Barr virus (EBV)-infected cells. Consistent with previous observations, sequence tags derived from miRNA loci in EBV and humans globally associate in approximately equivalent amounts with Ago1 and Ago2. Interestingly, additional sRNAs, which have not been registered as miRNAs, were associated with Ago1. Among them, some unique sequence tags derived from tandem loci in the human genome associate exclusively with Ago1 but not, or rarely, with Ago2. This is supported by the observation that the expression of the unique sRNAs in the cells is highly dependent on Ago1 proteins. When we knocked down Ago1, the expression of the Ago1-specific sRNAs decreased dramatically. Most importantly, the Ago1-specific sRNAs bound to mRNAs and regulated target genes and were dramatically upregulated, depending on the EBV life cycle. Therefore, even in mammals, the sorting mechanism in the Ago1–4 family is functional. Moreover, the existence of Ago1-specific sRNAs implies vital roles in some aspects of mammalian biology.

Myasthenia gravis after allogeneic bone marrow transplantation treated with mycophenolate mofetil monitored by peripheral blood OX40+ CD4+ T cells.

Abstract: A patient who developed myasthenia gravis (MG) 25 months after allogeneic bone marrow transplant was immunologically analyzed. OX40+CD4+ T cells in the peripheral blood prominently increased one month before the onset of MG. CD4/CD8 ratios, usually abnormally inverted in patients with chronic graft‐vs.‐host disease (cGVHD), showed pseudonormalization during the course of MG. We succeeded in uneventful rapid tapering of prednisolone (PSL) using mycophenolate mofetil (MMF). Monitoring of OX40+CD4+ T cells supported the tapering of PSL and MMF as a marker of cGVHD activity. This case suggested the utility of MMF and monitoring of OX40+CD4+ T cells in the management of cGVHD‐associated autoimmune diseases.