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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.

After the demonstration that surrogate JCkappa polypeptides could covalently bind mu heavy chain and upon the characterization of the Vkappa-like component of the kappa-like pre-B cell receptor, it became evident that germline transcription is not sterile. The present review discusses the concept of the alternative usage of kappa-like pre-B cell receptors and classical pre-B cell receptors utilizing the lambda-like surrogate light chain composed of lambda5 and VpreB. We propose that both kappa-like and lambda-like pre-B cell receptors work in concert in a fail-safe mechanism to promote light chain rearrangement, heavy chain allelic exclusion and B-lymphocyte maturation.

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.

By confining germline transcription as a byproduct of the mechanisms inherent to genetic rearrangements, the translation of respective mRNAs and their biological relevance might have been overlooked. Here we report the identification, cloning, and biochemical characterization of a human Vkappa-like protein that is encoded by a germline transcript. This surrogate protein assembles with the immunoglobulin mu heavy chain at the surface of B cell progenitors and precursors to form a kappa-like antigen receptor. These findings support the notion that germline transcription is not futile and stress the flexibility in eukaryotic gene usage and expression. In addition, the present study confirms the co-existence of surrogate lambda and kappa receptors that are proposed to work in concert to promote B lymphocyte maturation.

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 wild-type 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.

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 gain- and 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.

Opossums are animals that are naturally resistant to the proteolytic effects of Crotalid venoms. Opossums possess proteinase inhibitors in their sera that bind to and neutralize hemorrhagic and other proteolytic activity in many snake venoms. The proteinase inhibitors are not antibodies since they have different molecular weights (60kDa) and pI (4.2). The purpose of this study was to determine if opossums were capable of producing antibodies against venom and/or increasing the production of proteinase inhibitors (specifically antihemorrhagins). Five different venom immunization protocols were used to determine the effects of the venom in the opossums. The dosages ranged from 1mg of venom per immunization to 350mg/kg body weight of venom per immunization. The antihemorrhagic response was increased, but there is no evidence to suggest that an opossum can produce antibodies against venom. The lack of an antibody response is most likely due to the natural proteinase inhibitors clearing the venom from the opossum's body before an antibody response can occur.

To the editor: Identification of biomarkers for response to novel therapeutic agents is an essential step for the success of tailored treatments in cancer. Preliminary data from clinical trials indicate that inhibitors of the spleen tyrosine kinase (SYK) may be efficacious in subsets of diffuse

Snake venoms contain direct-acting fibrinolytic metalloproteinases (MMP) that could have important applications in medicine. Fibrinolytic enzymes isolated from venom can induce in vitro clot lysis by directly acting on a fibrin clot. The most ideal fibrinolytic enzyme would have high affinity for clots, dissolve clots directly without causing hemorrhage, and would not be neutralized in vivo by endogenous metalloproteinase inhibitors. The purpose of this study was to compare DEAE/HPLC venom profiles from Viperid snakes and identify fractions that contain fibrinolytic activity with no hemorrhagic activity and are not neutralized by animal sera. The sera selected were from four (Virginia opossum, Gray woodrat, Mexican ground squirrel, and Hispid cottonrat) animals known to neutralize hemorrhagic activity in snake venoms. Nineteen fractions from the Viperid venoms had fibrinolytic activity. Agkistrodon venom fractions contained the highest specific fibrinolytic activities. A. piscivorus leucostoma fraction 4 contained a high specific fibrinolytic activity, no hemorrhagic activity, and the fibrinolytic activity was not neutralized by the proteinase inhibitors of the four animal sera. A. contortrix laticinctus fraction 1 also had a high specific fibrinolytic activity and no hemorrhagic activity. However, the fibrinolytic activity was neutralized by Didelphis virginiana (Virginia opossum) serum.

Background LEA (late embryogenesis abundant) proteins encode conserved N-terminal mitochondrial signal domains and C-terminal (A/TAEKAK) motif repeats, long-presumed to confer cell resistance to stress and death cues. This prompted the hypothesis that LEA proteins are central to mitochondria mechanisms that connect bioenergetics with cell responses to stress and death signaling. In support of this hypothesis, recent studies have demonstrated that mammalian LEA protein PRELI can act as a biochemical hub, which upholds mitochondria energy metabolism, while concomitantly promoting B cell resistance to stress and induced death. Hence, it is important to define in vivo the physiological relevance of PRELI expression. Methods and findings Given the ubiquitous PRELI expression during mouse development, embryo lethality could be anticipated. Thus, conditional gene targeting was engineered by insertion of flanking loxP (flox)/Cre recognition sites on PRELI chromosome 13 (Chr 13) locus to abort its expression in a tissue-specific manner. After obtaining mouse lines with homozygous PRELI floxed alleles (PRELI(f/f)), the animals were crossed with CD19-driven Cre-recombinase transgenic mice to investigate whether PRELI inactivation could affect B-lymphocyte physiology and survival. Mice with homozygous B cell-specific PRELI deletion (CD19-Cre/Chr13 PRELI(-/-)) bred normally and did not show any signs of morbidity. Histopathology and flow cytometry analyses revealed that cell lineage identity, morphology, and viability were indistinguishable between wild type CD19-Cre/Chr13 PRELI(+/+) and CD19-Cre/Chr13 PRELI(-/-) deficient mice. Furthermore, B cell PRELI gene expression seemed unaffected by Chr13 PRELI gene targeting. However, identification of additional PRELI loci in mouse Chr1 and Chr5 provided an explanation for the paradox between LEA-dependent cytoprotection and the seemingly futile consequences of Chr 13 PRELI gene inactivation. Importantly, PRELI expression from spare gene loci appeared ample to surmount Chr 13 PRELI gene deficiency. Conclusions These findings suggest that PRELI is a vital LEA B cell protein with failsafe genetics.

Abstract 315 The mechanisms by which microRNA (miRNA) dysfunction contribute to the pathogenesis of diffuse large B-cell lymphoma (DLBCL) are not well established. MiRNAs are small non-protein coding RNAs that function by regulating the expression of target transcripts. Thus, to capture the physiologic and pathologic impact of these small regulatory molecules it is necessary to identify the genes that they inhibit. MicroRNA-155 (miR-155) is overexpressed in aggressive subsets of DLBCL, and its enforced expression in a transgenic, Eμ-miR-155, mouse model is associated with the development of lymphoblastic leukemia/high grade lymphoma. However, although a series of bona-fide miR-155 targets have been identified, it is still unclear how overexpression of this miR-155 contributes to lymphomagenesis. Starting from unbiased genome-wide approaches in DLBCL, we discovered that miR-155 directly binds to two sites in the 3'UTR of the SMAD5 gene, inhibiting its expression. Surprisingly, although SMAD5 activity is classically associated with signals transduced by bone morphogenic protein (BMP) family of cytokines, we found that in DLBCLs a non-canonical signaling module linking TGFβ1 to SMAD5 is also active. Furthermore, using kinase inhibitors specific to BMP or TGFβ receptors, we found that the type I TGFβ receptors, in particular ALK5, were essential for the activation of SMAD5 by TGFβ1 in DLBCL. These data suggested that the impact of miR-155 on the TGFβ pathway could be broader than previously appreciated, and abrogate the growth inhibitory effects of both BMP and TGFβ1 in DLBCL. To test this hypothesis, we generated DLBCL cell lines (Ly1, Ly18 and Ly19) ectopically expressing miR-155 and found that they became resistant to the cytostatic effects of TGFβ1 and BMPs. This blockade in growth inhibition was associated with an impaired cell cycle arrest and defective induction of p21. Next, to firmly establish the role of SMAD5 targeting in the miR-155-mediated disruption of the TGFβ signals, we created DLBCL cell lines stably expressing two independent SMAD5-shRNA constructs. Indeed, knockdown of SMAD5 in DLBCL limited the growth inhibitory effects of BMP and TGFβ1 and abrogated p21 induction, largely recapitulating the effects of miR-155 expression. In addition, using a xenograft model of DLBCL with in vivo luminescent imaging capabilities, we found that miR-155 overexpression yielded larger and more widespread lymphomas. Confirming our in vitro data, DLBCLs with stable SMAD5 knockdown were also more aggressive, indicating that SMAD5 downregulation phenocopies the effects of miR-155 in vivo. To gain further insights into the functional consequences of the miR-155/SMAD5 interplay, we used real-time RT-PCR to measure the transcriptional activation of several SMAD5 direct targets. In DLBCL cell lines ectopically expressing miR-155 or SMAD5 shRNA constructs, we found that the expression of the SMAD5 targets, ID1, ID2, SMAD6, SMAD7, BMPR2 and BMP6, was significantly diminished. These data further stressed the relevance of miR-155-mediated SMAD5 downregulation, and started to unveil the downstream components of SMAD5's tumor suppressive activities. Finally, we expanded our observations to primary tumors, and used western blotting to show that miR-155 overexpressing DLBCLs had significantly lower levels of SMAD5, and exhibited an impaired expression of its transcriptional targets. Together, our data instructed on miR-155 mediated lymphomagenesis, highlighted a hitherto unappreciated role of SMAD5 as a lymphoma suppressor gene, and defined a novel mechanism used by cancer cells to escape TGFβ growth inhibitory effects. Disclosures: No relevant conflicts of interest to declare.

The immune environment present after allogeneic hematopoietic stem cell transplantation (HSCT) contributes to the control of leukemia. Our laboratory has demonstrated in a murine model that vaccination of recipients after transplantation with recipient tumor vaccines does not exacerbate graft-versus-host disease but does induce meaningful graft-versus-tumor effects. We previously demonstrated that part of the reason for the lack of graft-versus-host disease from post-transplantation vaccination is due to gradual acquisition of tolerance or unresponsiveness to recipient immunodominant minor histocompatibility antigens that are ubiquitously expressed in the recipient. However, our prior studies have not critically addressed the question of whether a similar process of acquisition of unresponsiveness to or tolerance of antigens present on minimal residual disease also occurs. The present study tested the hypothesis that unresponsiveness to antigens present on minimal residual disease present at the time of HSCT would also occur. The answer to this question would have a significant effect on the potential efficacy of post-transplantation tumor vaccines. In a murine model of major histocompatibility complex matched, minor histocompatibility antigen mismatched HSCT (C3.SW female donors and C57BL/6 female recipients), we tested whether transplant recipients would acquire unresponsiveness to antigens present on small numbers of residual leukemia/lymphoma cells. We employed a male C57BL/6 lymphoid malignancy with an immunoglobulin/c-myc oncogene in these studies using as a model of tumor-restricted antigen the well-characterized male (HY) antigen system present only on the tumor but not present as ubiquitous minor antigens in the recipient. After HSCT, recipients did not mount immune responses to the ubiquitously distributed immunodominant recipient strain H7 minor histocompatibility antigen, but did retain the capacity to mount significant T cell responses to HY antigens present on small numbers of HY+ tumor cells present at transplantation. Additional studies using small numbers of nonmalignant recipient male B cells or dendritic cells as models of minimal residual disease also demonstrated that the transplant recipients retained their capacity to mount anti-HY T cell responses. After HSCT, recipients may retain the capacity to mount effective T cell responses to antigens present on minimal residual disease and still acquire relative tolerance to ubiquitously distributed immunodominant minor antigens that are related to graft-versus-host disease.