The mechanisms by which microRNA dysfunction contributes to the pathogenesis of diffuse large B cell lymphoma (DLBCL) are not well established. The identification of the genes and pathways directly targeted by these small regulatory RNAs is a critical step to advance this field. Using unbiased genome-wide approaches in DLBCL, we discovered that the oncogenic microRNA-155 (miR-155) directly targets the bone morphogenetic protein (BMP)-responsive transcriptional factor SMAD5. Surprisingly, we found that in DLBCL a noncanonical signaling module linking TGF-β1 signals to SMAD5 is also active. In agreement with these data, miR-155 overexpression rendered DLBCLs resistant to the growth-inhibitory effects of both TGF-β1 and BMPs, via defective induction of p21 and impaired cell cycle arrest. In confirmatory experiments, RNAi-based SMAD5 knockdown recapitulated in vitro and in vivo the effects miR-155 overexpression. Furthermore, in primary DLBCLs, miR-155 overexpression inhibited SMAD5 expression and disrupted its activity, as defined by individual and global analyses of its transcriptional targets. Together, our data helped explain miR-155 function, highlighted a hitherto unappreciated role of SMAD5 in lymphoma biology, and defined a unique mechanism used by cancer cells to escape TGF-β's growth-inhibitory effects.lymphoma | miR-155 | transforming growth factor β | B lymphocytes | bone morphogenetic protein S ignificant progress has been made recently in elucidating the molecular basis of diffuse large B cell lymphoma (DLBCL) (1). These studies were primarily centered on classic mRNA genes, whereas the role of microRNAs (miRNA) in lymphomagenesis remains to be fully appreciated. MiRNAs are non-protein-coding RNAs that function by regulating the expression of target transcripts (2). Thus, to capture the biologic impact of these regulatory molecules it is necessary to identify the genes that they inhibit. Micro-RNA-155 (miR-155) is overexpressed in aggressive DLBCLs (3, 4), and its aberrant expression in a transgenic, Eμ-miR-155, mouse model was associated with the development of lymphoblastic leukemia/high-grade lymphoma (5). However, although several bona fide miR-155 targets have been identified (6-10), it is unclear how their excessive down-regulation in DLBCLs that overexpress miR-155 contribute to lymphomagenesis. A direct way to address this problem is to propose that miR-155 physiologically targets B cell growth-suppressing pathways and that its abnormal expression in DLBCL curtails these inhibitory signals, contributing to an advantageous prosurvival behavior.Herein, we report that miR-155 binds to the 3′ UTR of the SMAD5 gene. Detailed characterization of this interaction showed that genetic modulation of miR-155 expression in DLBCL cell lines concomitantly changed SMAD5 levels. Although SMAD5 activity is classically associated with signals transduced by the BMP (bone morphogenetic protein) family of cytokines (11), we found that in DLBCL TGF-β1 also activated SMAD5. Thus, DLBCL cell lines engineered to expres...

Identification of rational therapeutic targets is an important strategy to improve the cure rate of diffuse large B-cell lymphoma (DLBCL). We previously showed that inhibition of the phosphodiesterase 4B (PDE4B) unleashes cyclic-AMP (cAMP) inhibitory effects toward the PI3K/AKT pathway and induces apoptosis. These data raised important considerations as to which upstream regulators mediate cAMP inhibition of PI3K/AKT, and how identifying this signaling route could be translated into clinical initiatives. We found that in normal and malignant B cells, cAMP potently inhibit the phosphorylation and activity of the tyrosine kinase SYK. Using genetic models of gainand loss-of-function, we demonstrated the essential role for PDE4B in controlling these effects in DLBCL. Furthermore, we used a constitutively active SYK mutant to confirm its central role in transducing cAMP effects to PI3K/AKT. Importantly, given SYK credentials as a therapeutic target in B-cell tumors, we explored the role of PDE4B in these responses. In multiple DLBCL models, we found that genetically, hence specifically, inhibiting PDE4B expression significantly improved the efficacy of SYK inhibitors. Our data defined a hitherto unknown role for cAMP in negatively regulating SYK and indicate that combined inhibition of PDE4B and SYK should be actively pursued. (Blood. 2009;113: 6153-6160)

Proteins containing the late embryogenesis abundant (LEA) motif comprise a conserved family, postulated to act as cell protectors. However, their function and mechanisms of action remain unclear. Here we show that PRELI, a mammalian LEA-containing homolog of yeast Ups1p, can associate with dynamin-like GTPase Optic Atrophy-1 (OPA1) and contribute to the maintenance of mitochondrial morphology. Accordingly, PRELI can uphold mitochondrial membrane potential (ΔΨm) and enhance respiratory chain (RC) function, shown by its capacity to induce complex-I/NADH dehydrogenase and ATP synthase expression, increase oxygen consumption and reduce reactive oxygen species (ROS) production. PRELI can also inhibit cell death induced by STS, TNF-α or UV irradiation. Moreover, in vitro and in vivo dominant-negative overexpression of mutant PRELI/LEA− (lacking the LEA motif) and transient in vitro PRELI-specific knockdown can render lymphocytes vulnerable to apoptosis, cause mouse embryo lethality and revert the resistance of lymphoma cells to induced death. Collectively, these data support the long-presumed notion of LEA protein-dependent mechanisms of cytoprotection and suggest that PRELI interacts with OPA1 to maintain mitochondria structures intact, sustain balanced ion−/proton+ gradients, promote oxidative phosphorylation reactions, regulate pro- and antiapoptotic protein traffic and enable cell responses to induced death. These findings may help to understand how bioenergetics is mechanistically connected with cell survival cues.

Gene expression can be regulated by chromatin modifiers, transcription factors and proteins that modulate DNA architecture. Among the latter, AT-hook transcription factors have emerged as multifaceted regulators that can activate or repress broad A/T-rich gene networks. Thus, alterations of AT-hook genes could affect the transcription of multiple genes causing global cell dysfunction. Here we report that targeted deletions of mouse AKNA, a hypothetical AT-hook-like transcription factor, sensitize mice to pathogen-induced inflammation and cause sudden neonatal death. Compared with wild-type littermates, AKNA KO mice appeared weak, failed to thrive and most died by postnatal day 10. Systemic inflammation, predominantly in the lungs, was accompanied by enhanced leukocyte infiltration and alveolar destruction. Cytologic, immunohistochemical and molecular analyses revealed CD11b + Gr1 + neutrophils as major tissue infiltrators, neutrophilic granule protein, cathelin-related antimicrobial peptide and S100A8/9 as neutrophil-specific chemoattracting factors, interleukin-1β and interferon-γ as proinflammatory mediators, and matrix metalloprotease 9 as a plausible proteolytic trigger of alveolar damage. AKNA KO bone marrow transplants in wildtype recipients reproduced the severe pathogen-induced reactions and confirmed the involvement of neutrophils in acute inflammation. Moreover, promoter/reporter experiments showed that AKNA could act as a gene repressor. Our results support the concept of coordinated pathway-specific gene regulation functions modulating the intensity of inflammatory responses, reveal neutrophils as prominent mediators of acute inflammation and suggest mechanisms underlying the triggering of acute and potentially fatal immune reactions.

We previously showed that the human AKNA gene encodes an AT-hook transcription factor that regulates the expression of costimulatory cell surface molecules on lymphocytes. However, AKNA cDNA probes hybridize with multiple transcripts, suggesting either the existence of other homologous genes or a complex regulation operating on a single gene. Here we report evidence for the latter, as we find that AKNA is encoded by a single gene that spans a 61-kb locus of 24 exons on the fragile FRA9E region of human chromosome 9q32. This gene gives rise to at least nine distinct transcripts, most of which are expressed in a tissue-specific manner in lymphoid organs. Many of the AKNA transcripts originate from alternative splicing; others appear to derive from differential polyadenylation and promoter usage. The alternative AKNA transcripts are predicted to encode overlapping protein isoforms, some of which (p70 and p100) are readily detectable using a polyclonal anti-AKNA antisera that we generated. We also find that AKNA PEST-dependent cleavage into p50 polypeptides is targeted to mature B cells and appears to be required for CD40 upregulation. The unusual capacity of the AKNA gene to generate multiple transcripts and proteins may reflect its functional diversity, and it may also provide a fail-safe mechanism that preserves AKNA expression.