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Although midnolin has been studied for over 20 years, its biological roles in vivo remain largely unknown, especially due to the lack of a functional animal model. Indeed, given our recent discovery that knockdown of midnolin suppresses liver cancer cell tumorigenicity and that this anti-tumorigenic effect is associated with modulation of lipid metabolism, we hypothesized that knockout of midnolin in vivo could potentially protect from nonalcoholic fatty liver disease (NAFLD) which has become the most common cause of chronic liver disease in the Western world. Accordingly, in the present study, we have developed and now report on the first functional global midnolin knockout mouse model. While the overwhelming majority of global homozygous midnolin knockout mice demonstrated embryonic lethality, heterozygous knockout mice were observed to be similar to wild-type mice in their viability and were used to determine the effect of reduced midnolin expression on NAFLD. We found that global heterozygous midnolin knockout attenuated the severity of NAFLD in mice fed a Western-style diet, high in fat, cholesterol, and fructose, and this attenuation in disease was associated with significantly reduced levels of large lipid droplets, hepatic free cholesterol, and serum LDL, with significantly differential gene expression involved in cholesterol/lipid metabolism. Collectively, our results support a role for midnolin in regulating cholesterol/lipid metabolism in the liver. Thus, midnolin may represent a novel therapeutic target for NAFLD. Finally, our observation that midnolin was essential for survival underscores the broad importance of this gene beyond its role in liver biology.

XLS file - 204K, Genomic regions enriched in MLLN, AF4C, H3K79me2 and the common of the three ChIP-seq in RS4;11 cells

XLS file - 330K, Genes included in BCR-ABL1, E2A-PBX1 or MLLr DNA methylation signatures vs. normal pre-B cells

XLS file - 60K, Characteristics of the 215 E2993 B-ALL patients and the 12 normal bone marrow samples (excel file)

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XLS file - 74K, DNA methylation and gene expression signature of BCR-ABL1-positive B-ALL

Hepatocellular carcinoma (HCC) ranks worldwide as one of the most lethal cancers. In spite of the vast existing knowledge about HCC, the pathogenesis of HCC is not completely understood. Discovery of novel genes that contribute to HCC pathogenesis will provide new insights for better understanding and treating HCC. The relatively obscure gene midnolin has been studied for over two decades; however, its biological roles are largely unknown. Our study is the first to demonstrate the functional significance of midnolin in HCC/cancer: Midnolin expression correlates with poor prognosis in HCC patients, and suppression of midnolin severely inhibits tumorigenicity of HCC cells in vitro and in mice and disrupts retinoic acid/lipid metabolism in these cells.

Background: Although cyclic AMP-response element binding protein-binding protein (CBP)/β-catenin signaling is known to promote proliferation and fibrosis in various organ systems, its role in the activation of pancreatic stellate cells (PSCs), the key effector cells of desmoplasia in pancreatic cancer and fibrosis in chronic pancreatitis, is largely unknown. Methods: To investigate the role of the CBP/β-catenin signaling pathway in the activation of PSCs, we have treated mouse and human PSCs with the small molecule specific CBP/β-catenin antagonist ICG-001 and examined the effects of treatment on parameters of activation. Results: We report for the first time that CBP/β-catenin antagonism suppresses activation of PSCs as evidenced by their decreased proliferation, down-regulation of “activation” markers, e.g., α-smooth muscle actin (α-SMA/Acta2), collagen type I alpha 1 (Col1a1), Prolyl 4-hydroxylase, and Survivin, up-regulation of peroxisome proliferator activated receptor gamma (Ppar-γ) which is associated with quiescence, and reduced migration; additionally, CBP/β-catenin antagonism also suppresses PSC-induced migration of cancer cells. Conclusion: CBP/β-catenin antagonism represents a novel therapeutic strategy for suppressing PSC activation and may be effective at countering PSC promotion of pancreatic cancer.

Summary Adenine N6 methylation in DNA (6mA) is widespread among bacteria and phage and is detected in mammalian genomes, where its function is largely unexplored. Here we show that 6mA deposition and removal are catalyzed by the Mettl4 methyltransferase and Alkbh4 dioxygenase, respectively, and that 6mA accumulation in genic elements corresponds with transcriptional silencing. Inactivation of murine Mettl4 depletes 6mA and causes sublethality and craniofacial dysmorphism in incross progeny. We identify distinct 6mA sensor domains of prokaryotic origin within the MPND deubiquitinase and ASXL1, a component of the Polycomb repressive deubiquitinase (PR-DUB) complex, both of which act to remove monoubiquitin from histone H2A (H2A-K119Ub), a repressive mark. Deposition of 6mA by Mettl4 triggers the proteolytic destruction of both sensor proteins, preserving genome-wide H2A-K119Ub levels. Expression of the bacterial 6mA methyltransferase Dam, in contrast, fails to destroy either sensor. These findings uncover a native, adversarial 6mA network architecture that preserves Polycomb silencing., Graphical Abstract, Highlights • 6mA deposition and erasure by mammalian Mettl4 and Alkbh4, respectively • Mettl4-deficient mice display craniofacial dysmorphism • 6mA triggers proteolysis of its cognate sensor proteins ASXL1 and MPND • Adversarial 6mA network architecture preserves Polycomb silencing, Kweon et al. reveal the molecular infrastructure of a mammalian DNA N6-methyladenine (6mA) methylation, erasure, and sensing system. Deposition of 6mA by the Mettl4 methyltransferase triggers the proteolytic destruction of its cognate sensor proteins, the histone H2A deubiquitinases ASXL1 and MPND, thus preserving Polycomb gene silencing.

© 2015 Nature America, Inc. All rights reserved. Childhood acute lymphoblastic leukemia (ALL) can often be traced to a pre-leukemic clone carrying a prenatal genetic lesion. Postnatally acquired mutations then drive clonal evolution toward overt leukemia. The enzymes RAG1-RAG2 and AID, which diversify immunoglobulin-encoding genes, are strictly segregated in developing cells during B lymphopoiesis and peripheral mature B cells, respectively. Here we identified small pre-BII cells as a natural subset with increased genetic vulnerability owing to concurrent activation of these enzymes. Consistent with epidemiological findings on childhood ALL etiology, susceptibility to genetic lesions during B lymphopoiesis at the transition from the large pre-BII cell stage to the small pre-BII cell stage was exacerbated by abnormal cytokine signaling and repetitive inflammatory stimuli. We demonstrated that AID and RAG1-RAG2 drove leukemic clonal evolution with repeated exposure to inflammatory stimuli, paralleling chronic infections in childhood.

Background & Aims Alcohol is a primary cause of liver disease and an important co-morbidity factor in other causes of liver disease. A common feature of progressive liver disease is fibrosis, which results from the net deposition of fibril-forming extracellular matrix (ECM). The hepatic stellate cell (HSC) is widely considered to be the major cellular source of fibrotic ECM. We determined if HSCs are responsive to direct stimulation by alcohol. Methods HSCs undergoing transdifferentiation were incubated with ethanol and expression of fibrogenic genes and epigenetic regulators was measured. Mechanisms responsible for recorded changes were investigated using ChIP-Seq and bioinformatics analysis. Ethanol induced changes were confirmed using HSCs isolated from a mouse alcohol model and from ALD patient's liver and through precision cut liver slices. Results HSCs responded to ethanol exposure by increasing profibrogenic and ECM gene expression including elastin. Ethanol induced an altered expression of multiple epigenetic regulators, indicative of a potential to modulate chromatin structure during HSC transdifferentiation. MLL1, a histone 3 lysine 4 (H3K4) methyltransferase, was induced by ethanol and recruited to the elastin gene promoter where it was associated with enriched H3K4me3, a mark of active chromatin. Chromatin immunoprecipitation sequencing (ChIPseq) revealed that ethanol has broad effects on the HSC epigenome and identified 41 gene loci at which both MML1 and its H3K4me3 mark were enriched in response to ethanol. Conclusions Ethanol directly influences HSC transdifferentiation by stimulating global changes in chromatin structure, resulting in the increased expression of ECM proteins. The ability of alcohol to remodel the epigenome during HSC transdifferentiation provides mechanisms for it to act as a co-morbidity factor in liver disease.

Summary: Enzymatic oxidation of 5-methylcytosine (5mC) in DNA by the Tet dioxygenases reprograms genome function in embryogenesis and postnatal development. Tet-oxidized derivatives of 5mC such as 5-hydroxymethylcytosine (5hmC) act as transient intermediates in DNA demethylation or persist as durable marks, yet how these alternative fates are specified at individual CpGs is not understood. Here, we report that the SOS response-associated peptidase (SRAP) domain protein Srap1, the mammalian ortholog of an ancient protein superfamily associated with DNA damage response operons in bacteria, binds to Tet-oxidized forms of 5mC in DNA and catalyzes turnover of these bases to unmodified cytosine by an autopeptidase-coupled nuclease. Biallelic inactivation of murine Srap1 causes embryonic sublethality associated with widespread accumulation of ectopic 5hmC. These findings establish a function for a class of DNA base modification-selective nucleases and position Srap1 as a determinant of 5mC demethylation trajectories during mammalian embryonic development. : Kweon et al. uncover a function for the SRAP domain, which couples autoproteolytic cleavage to activation of a nuclease selective for DNA containing Tet-oxidized derivatives of 5-methylcytosine. These findings reveal a mechanism for targeted erasure of DNA methylation via the stepwise enzymatic actions of Tet and SRAP. Keywords: DNA methylation, Tet, SRAP, endonuclease, autopeptidase, embryonic stem cell

Background & Aims Stearoyl-CoA desaturase (SCD) synthesizes monounsaturated fatty acids (MUFAs) and has been associated with the development of metabolic syndrome, tumorigenesis, and stem cell characteristics. We investigated whether and how SCD promotes liver fibrosis and tumor development in mice. Methods Rodent primary hepatic stellate cells (HSCs), mouse liver tumor-initiating stem cell-like cells (TICs), and human hepatocellular carcinoma (HCC) cell lines were exposed to Wnt signaling inhibitors and changes in gene expression patterns were analyzed. We assessed the functions of SCD by pharmacologic and conditional genetic manipulation in mice with hepatotoxic or cholestatic induction of liver fibrosis, orthotopic transplants of TICs, or liver tumors induced by administration of diethyl nitrosamine. We performed bioinformatic analyses of SCD expression in HCC vs nontumor liver samples collected from patients, and correlated levels with HCC stage and patient mortality. We performed nano-bead pull-down assays, liquid chromatography–mass spectrometry, computational modeling, and ribonucleoprotein immunoprecipitation analyses to identify MUFA-interacting proteins. We examined the effects of SCD inhibition on Wnt signaling, including the expression and stability of low-density lipoprotein–receptor–related proteins 5 and 6 (LRP5 and LRP6), by immunoblot and quantitative polymerase chain reaction analyses. Results SCD was overexpressed in activated HSC and HCC cells from patients; levels of SCD messenger RNA (mRNA) correlated with HCC stage and patient survival time. In rodent HSCs and TICs, the Wnt effector β-catenin increased sterol regulatory element binding protein 1–dependent transcription of Scd , and β-catenin in return was stabilized by MUFAs generated by SCD. This loop required MUFA inhibition of binding of Ras–related nuclear protein 1 (Ran1) to transportin 1 and reduced nuclear import of elav-like protein 1 (HuR), increasing cytosolic levels of HuR and HuR–mediated stabilization of mRNAs encoding LRP5 and LRP6. Genetic disruption of Scd and pharmacologic inhibitors of SCD reduced HSC activation and TIC self-renewal and attenuated liver fibrosis and tumorigenesis in mice. Conditional disruption of Scd2 in activated HSCs prevented growth of tumors from TICs and reduced the formation of diethyl nitrosamine–induced liver tumors in mice. Conclusions In rodent HSCs and TICs, we found SCD expression to be regulated by Wnt-β-catenin signaling, and MUFAs produced by SCD provided a forward loop to amplify Wnt signaling via stabilization of Lrp5 and Lrp6 mRNAs, contributing to liver fibrosis and tumor growth. SCD expressed by HSCs promoted liver tumor development in mice. Components of the identified loop linking HSCs and TICs might be therapeutic targets for liver fibrosis and tumors.

PPAR-γ is essential for differentiation of hepatic stellate cells (HSC), and its loss due to epigenetic repression by methyl-CpG binding protein 2 (MeCP2) causes HSC myofibroblastic activation mediated in part via Wnt pathway, the key cellular event in liver fibrosis. Decreased miR-132 was previously proposed to promote MeCP2 protein translation for Ppar-γ repression in activated HSC (aHSC). The present study aimed to test this notion and to better understand the mechanisms of MeCP2 upregulation in aHSC. MeCP2 protein is increased on day 3 to 7 as HSC become activated in primary culture on plastic, but this is accompanied by increased but not reduced miR-132 or miR-212 which is also expected to target MeCP2 due to its similar sequence with miR-132. The levels of these mRNAs are decreased 40~50% in aHSCs isolated from experimental cholestatic liver fibrosis but increased 6-8 fold in aHSC from hepatotoxic liver fibrosis in rats. Suppression of either or both of miR132 and miR212 with specific anti-miRNA oligonucleotides (anti-oligo), does not affect MeCP2 protein levels in aHSCs. The Wnt antagonist FJ9 which inhibits HSC activation, increases miR-132/miR-212, reduces MeCP2 and its enrichment at 5' Ppar-γ promoter, and restores Ppar-γ expression but the anti-oligo do not prevent Ppar-γ upregulation. The pan-NADPH oxidase (NOX) inhibitor diphenyleneiodonium (DPI) also reduces both MeCP2 and stabilized non-(S33/S37/Thr41)-phospho β-catenin and reverts aHSC to quiescent cells but do not affect miR-132/miR-212 levels. Wnt antagonism with FJ9 increases MeCP2 protein degradation in cultured HSC, and FJ9-mediated loss of MeCP2 is rescued by leupeptin but not by proteasome and lysozome inhibitors. In conclusion, canonical Wnt pathway increases MeCP2 protein due to protein stability which in turn represses Ppar-γ and activates HSC.

BCL6 protects germinal center (GC) B cells against DNA damage–induced apoptosis during somatic hypermutation and class-switch recombination. Although expression of BCL6 was not found in early IL-7–dependent B cell precursors, we report that IL-7Rα–Stat5 signaling negatively regulates BCL6. Upon productive VH-DJH gene rearrangement and expression of a μ heavy chain, however, activation of pre–B cell receptor signaling strongly induces BCL6 expression, whereas IL-7Rα–Stat5 signaling is attenuated. At the transition from IL-7–dependent to –independent stages of B cell development, BCL6 is activated, reaches expression levels resembling those in GC B cells, and protects pre–B cells from DNA damage–induced apoptosis during immunoglobulin (Ig) light chain gene recombination. In the absence of BCL6, DNA breaks during Ig light chain gene rearrangement lead to excessive up-regulation of Arf and p53. As a consequence, the pool of new bone marrow immature B cells is markedly reduced in size and clonal diversity. We conclude that negative regulation of Arf by BCL6 is required for pre–B cell self-renewal and the formation of a diverse polyclonal B cell repertoire.

In review of the past studies on NF-kappaB regulation, most of them have focused on investigating how NF-kappaB is activated by a single inducer at a time. Given the fact that, in mixed bacterial infections in vivo, multiple inflammation inducers, including both nontypeable Haemophilus influenzae (NTHi) and Streptococcus pneumoniae, are present simultaneously, a key issue that has yet to be addressed is whether NTHi and S. pneumoniae simultaneously activate NF-kappaB and the subsequent inflammatory response in a synergistic manner. Here, we show that NTHi and S. pneumoniae synergistically induce NF-kappaB-dependent inflammatory response via activation of multiple signaling pathways in vitro and in vivo. The classical IKKbeta-IkappaBalpha and p38 MAPK pathways are involved in synergistic activation of NF-kappaB via two distinct mechanisms, p65 nuclear translocation-dependent and -independent mechanisms. Moreover, casein kinase 2 (CK2) is involved in synergistic induction of NF-kappaB via a mechanism dependent on phosphorylation of p65 at both Ser536 and Ser276 sites. These studies bring new insights into the molecular mechanisms underlying the NF-kappaB-dependent inflammatory response in polymicrobial infections and may lead to development of novel therapeutic strategies for modulating inflammation in mixed infections for patients with otitis media and chronic obstructive pulmonary diseases.

Activated hepatic stellate cells (HSCs) are primarily responsible for the genesis of liver fibrosis which promotes liver tumor development. Stearoyl-CoA desaturase (SCD) which synthesizes monounsaturated fatty acid (MUFA such as oleic acid or palmitoleic acid), is implicated in metabolic syndrome, tumorigenesis and stemness. Indeed, we demonstrate SCD upregulation in both HSCs and liver tumor cells in patients. Further, SCD correlates with the advancing grade of HCC and mortality in patients. However, its causality in tumorigenesis and mesenchyme-tumor crosstalk, and underlying mechanisms, are unknown. The present study aimed to determine whether and how SCD promotes and links liver fibrosis and cancer. In both HSCs and liver tumor-initiating stem cell-like cells (TIC), we reveal that the Wnt effector β-catenin is a potent co-activator for SREBP-1-dependent Scd transcription as demonstrated by promoter-reporter assay and re-ChIP analysis. We also discover β-catenin which drives SCD expression is in turn stabilized by SCD-derived MUFA. To identify molecular targets of MUFA mediating this SCD-β-catenin positive loop, we performed MUFA-nano-beads pull-down assay with TIC lysate, ESI-MS based identification of MUFA interacting proteins, computational docking analysis, co-IP and ribonucleoprotein-IP. These analyses reveal that MUFA is essential for LRP5/6 expression: MUFA interferes Transportin-1 and Ran-1 interaction, resulting in suppressed nuclear import of the mRNA binding protein HuR, HuR cytoplasmic accumulation, and increased HuR binding to and stabilization of Wnt co-receptor Lrp5/6 mRNA. As such, genetic or pharmacologic SCD inhibition reduces cytosolic HuR, LRP5/6 expression, β-catenin stabilization, HSC activation and TIC self-renewal and attenuates liver fibrosis and tumorigenesis in mice. Moreover, conditional genetic ablation of Scd2 in HSCs not only abrogates HSC-mediated promotion of TIC-derived tumorigenesis in a xenograft mouse model but also inhibits DEN-induced liver tumor development in Col1a1-Cre; Scd2flox/flox mice (tumor volume reduced to 21.5 + 7.9 vs. 360.9 + 193 mm3 in Scd2flox/flox mice; tumor multiplicity decreased to 18 + 5.4 vs. 45 + 8.4 in Scd2flox/flox mice, both p Citation Format: Keane Lai, Soo-Mi Kweon, Feng Chi, Edward Hwang, Raymond Wu, Yasuaki Kabe, Ramachandran Murali, Lopa Mishra, James Ntambi, Hidekazu Tsukamoto. Novel Wnt-SCD-LRP5/6 pathway linking liver fibrosis to cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4331. doi:10.1158/1538-7445.AM2017-4331

Genetic lesions such as BCR–ABL1, E2A–PBX1, and MLL rearrangements (MLLr) are associated with unfavorable outcomes in adult B-cell precursor acute lymphoblastic leukemia (B-ALL). Leukemia oncoproteins may directly or indirectly disrupt cytosine methylation patterning to mediate the malignant phenotype. We postulated that DNA methylation signatures in these aggressive B-ALLs would point toward disease mechanisms and useful biomarkers and therapeutic targets. We therefore conducted DNA methylation and gene expression profiling on a cohort of 215 adult patients with B-ALL enrolled in a single phase III clinical trial (ECOG E2993) and normal control B cells. In BCR–ABL1-positive B-ALLs, aberrant cytosine methylation patterning centered around a cytokine network defined by hypomethylation and overexpression of IL2RA(CD25). The E2993 trial clinical data showed that CD25 expression was strongly associated with a poor outcome in patients with ALL regardless of BCR–ABL1 status, suggesting CD25 as a novel prognostic biomarker for risk stratification in B-ALLs. In E2A–PBX1-positive B-ALLs, aberrant DNA methylation patterning was strongly associated with direct fusion protein binding as shown by the E2A–PBX1 chromatin immunoprecipitation (ChIP) sequencing (ChIP-seq), suggesting that E2A–PBX1 fusion protein directly remodels the epigenome to impose an aggressive B-ALL phenotype. MLLr B-ALL featured prominent cytosine hypomethylation, which was linked with MLL fusion protein binding, H3K79 dimethylation, and transcriptional upregulation, affecting a set of known and newly identified MLL fusion direct targets with oncogenic activity such as FLT3 and BCL6. Notably, BCL6 blockade or loss of function suppressed proliferation and survival of MLLr leukemia cells, suggesting BCL6-targeted therapy as a new therapeutic strategy for MLLr B-ALLs. Significance: We conducted the first integrative epigenomic study in adult B-ALLs, as a correlative study to the ECOG E2993 phase III clinical trial. This study links for the first time the direct actions of oncogenic fusion proteins with disruption of epigenetic regulation mediated by cytosine methylation. We identify a novel clinically actionable biomarker in B-ALLs: IL2RA(CD25), which is linked with BCR–ABL1 and an inflammatory signaling network associated with chemotherapy resistance. We show that BCL6 is a novel MLL fusion protein target that is required to maintain the proliferation and survival of primary human adult MLLr cells and provide the basis for a clinical trial with BCL6 inhibitors for patients with MLLr. Cancer Discov; 2(11); 1004–23. ©2012 AACR. Read the Commentary on this article by Cimmino and Aifantis, p. 976. This article is highlighted in the In This Issue feature, p. 961

Tyrosine kinase inhibitors (TKIs) are widely used to treat patients with leukaemia driven by BCR-ABL1 (ref. 1) and other oncogenic tyrosine kinases. Recent efforts have focused on developing more potent TKIs that also inhibit mutant tyrosine kinases. However, even effective TKIs typically fail to eradicate leukaemia-initiating cells (LICs), which often cause recurrence of leukaemia after initially successful treatment. Here we report the discovery of a novel mechanism of drug resistance, which is based on protective feedback signalling of leukaemia cells in response to treatment with TKI. We identify BCL6 as a central component of this drug-resistance pathway and demonstrate that targeted inhibition of BCL6 leads to eradication of drug-resistant and leukaemia-initiating subclones.

Background RhoGDI proteins are important regulators of the small GTPase Rac, because they shuttle Rac from the cytoplasm to membranes and also protect Rac from activation, deactivation and degradation. How the binding and release of Rac from RhoGDI is regulated is not precisely understood. Results We report that the non-receptor tyrosine kinase Fer is able to phosphorylate RhoGDIα and form a direct protein complex with it. This interaction is mediated by the C-terminal end of RhoGDIα. Activation of Fer by reactive oxygen species caused increased phosphorylation of RhoGDIα and pervanadate treatment further augmented this. Tyrosine phosphorylation of RhoGDIα by Fer prevented subsequent binding of Rac to RhoGDIα, but once a RhoGDIα-Rac complex was formed, the Fer kinase was not able to cause Rac release through tyrosine phosphorylation of preformed RhoGDIα-Rac complexes. Conclusions These results identify tyrosine phosphorylation of RhoGDIα by Fer as a mechanism to regulate binding of RhoGDIα to Rac.

Human epithelial mucin, the major glycoprotein component of mucus, plays a critical role in host innate defense response against invading microbes by facilitating the mucociliary clearance. Excess mucin production, however, overwhelms the mucociliary clearance, resulting in not only defective mucosal defense but also conductive hearing loss in the middle ear and mucus obstruction in the airway. Indeed, mucus overproduction is a hall-mark of otitis media (OM) and chronic obstructive pulmonary diseases (COPD). Thus, tight regulation of mucin production plays an important role in maintaining an appropriate balance between beneficial and detrimental outcomes. We previously reported that Streptococcus pneumoniae (S. pneumoniae) up-regulates MUC5AC mucin expression via a positive MAPK ERK1/2 and a negative JNK1/2 signaling pathway. However, the signaling components including the up-stream activators and the down-stream transcription factors involved in these two path-ways remain largely unknown. In the present study, we showed that positive regulation of MUC5AC mucin expression by ERK1/2 is dependent on Ras-Raf-1 signaling pathway, whereas the negative regulation of MUC5AC expression by JNK1/2 is dependent on MEKK3. Moreover, transcriptional factor AP-1 acts as a key regulator for both of the positive and negative regulation of MUC5AC mucin expression as evidenced by mutagenesis analysis of two AP-1 sites in the promoter region of human MUC5AC mucin gene. Ras-Raf1-ERK1/2-dependent AP-1 activation positively regulates MUC5AC mucin induction by S. pneumoniae, whereas MEKK3-JNK1/2-dependent AP-1 activation negatively regulates it. Therefore, our data unveiled a novel signaling mechanism underlying the tight regulation of MUC5AC mucin induction by S. pneumoniae and may lead to the development of new therapeutic strategy for reducing mucus overproduction in both OM and COPD.

Transforming growth factor-beta (TGF-beta) family members are multifunctional growth factors involved in regulating diverse biological processes. Despite the critical role for TGF-beta in regulating cell proliferation, differentiation, migration and development, its role in regulating NF-kappaB-dependent inflammatory response still remains unclear. Here, we show that TGF-beta1 induces acetylation of NF-kappaB p65 subunit to synergistically enhance bacterium nontypeable Haemophilus influenzae-induced NF-kappaB activation and inflammatory response in vitro and in vivo. The TGF-beta1-induced acetylation of p65 is mediated via a Smad3/4-PKA-p300-dependent signaling pathway. Acetylation of p65 at lysine 221 by TGF-beta1 is critical for synergistic enhancement of bacteria-induced DNA-binding activity, NF-kappaB activation, NF-kappaB-dependent transcription of TNF-alpha and IL-1beta and interstitial polymorphonuclear neutrophil infiltration in vitro and in vivo. These studies provide new insights into the novel regulation of NF-kappaB by TGF-beta signaling.

In review of the past studies on NF-kappaB regulation, most of them have focused on investigating how NF-kappaB is activated by a single inducer at a time. Given the fact that, in mixed bacterial infections in vivo, multiple inflammation inducers, including both nontypeable Haemophilus influenzae (NTHi) and Streptococcus pneumoniae, are present simultaneously, a key issue that has yet to be addressed is whether NTHi and S. pneumoniae simultaneously activate NF-kappaB and the subsequent inflammatory response in a synergistic manner. Here, we show that NTHi and S. pneumoniae synergistically induce NF-kappaB-dependent inflammatory response via activation of multiple signaling pathways in vitro and in vivo. The classical IKKbeta-IkappaBalpha and p38 MAPK pathways are involved in synergistic activation of NF-kappaB via two distinct mechanisms, p65 nuclear translocation-dependent and -independent mechanisms. Moreover, casein kinase 2 (CK2) is involved in synergistic induction of NF-kappaB via a mechanism dependent on phosphorylation of p65 at both Ser536 and Ser276 sites. These studies bring new insights into the molecular mechanisms underlying the NF-kappaB-dependent inflammatory response in polymicrobial infections and may lead to development of novel therapeutic strategies for modulating inflammation in mixed infections for patients with otitis media and chronic obstructive pulmonary diseases.

Background and hypothesis: Childhood pre-B acute lymphoblastic leukemia (ALL) can frequently be retraced to a pre-leukemic clone carrying a prenatally acquired genetic lesion (e.g. ETV6-RUNX1gene rearrangement). After birth, pre-leukemic clones can acquire secondary mutations and, hence, evolve towards overt leukemia. While this concept is well established, the mechanism(s) driving clonal evolution are not known. Epidemiological findings hint to a role of delayed childhood infections and chronic inflammation as etiologic factors of childhood ALL, but do not illuminate mechanism of clonal evolution of pre-leukemic cells. In this study, we demonstrate that cooperation between the AID cytosine deaminase and the RAG1/RAG2 V(D)J recombinase promotes acquisition of secondary genetic lesions that promote progress of pre-leukemic B cell precursors towards full-blown leukemia. Results: The enzymatic activity of RAG1/RAG2 (VDJ recombination) and AID (somatic hypermutation, class-switch recombination) are strictly segregated to early and late stages of B cell development, respectively. While RAG1 and RAG2 are actively expressed at stages of early B cell development (bone marrow and fetal liver) that give rise to pre-B ALL, little is known about the function of AID in early B-lymphopoesis. As the involvement of AID in pre-B leukemic clonal evolution is incumbent on its expression during early stages of B-lymphopoesis, we tested CD19+ pre-B cells isolated from human bone marrow (BM) for indicators of AID activity, namely, somatic hypermutation (SHM) and class switch recombination (CSR). Interestingly, most pre-B cell clones carry rearranged Ig VH region genes that are mutated at low levels (average mutation frequency 26 x 10-3 bp). Likewise, pre-B cells isolated from fetal liver tissues (three donors; 10-19 weeks of gestation) carried Ig VH region genes mutated at low levels (average mutation frequency 14 x 10-3 bp). In addition, about one third of fetal liver pre-B cells had undergone CSR to Cγ3, Cγ1 and Cα regions. These findings highlight the previously unknown function of AID in two important sites of early human B-lymphopoesis. Based on these results, we hypothesized that a specific B cell subset during early pro- and pre-B cell differentiation can concomitantly express both AID and the RAGs and, hence, would be particularly susceptible to clonal evolution of cells that carry a pre-leukemic lesion. Our subsequent studies identified late pre-B cells (Fraction D) as a natural subset of increased genetic vulnerability. Late pre-B cells downregulate IL7 receptor/Stat5 signaling, which enables expression of RAG1 and RAG2 and immunoglobulin light chain gene rearrangement. Loss of IL7 receptor/Stat5 signaling also removes an important safeguard against premature expression of AID. Therefore, late pre-B cells are poised to express AID at high levels in response to inflammatory stimuli (e.g. LPS) in concurrence with RAG1 and RAG2. Studying clonal evolution of patient-derived pre-B ALL cells, we found evidence for concomitant AID and RAG1/RAG2 activity. Further studying a genetic mouse model for pre-leukemic pre-B cells carrying ETV6-RUNX1, we found that repeated exposure to LPS can cause overt leukemia but not in the absence of either AID or Rag1. Additionally, whole exome sequencing of human B cell clones that were engineered to express AID, RAG1/RAG2 alone or in combination revealed that concurrent expression of AID with RAG1/RAG2 dramatically increased the frequency of structural chromosomal lesions. Conclusion: Consistent with epidemiological findings on the etiology of childhood ALL, we conclude that reduced cytokine signaling (here, IL7R) in late pre-B cells renders pre-leukemic clones distinctively vulnerable to genetic lesions that can be acquired in the context of repeated exposure to inflammatory stimuli (e.g. chronic and recurrent infections during childhood). Our results support a role for AID and RAGs cooperation for the generation of secondary lesions in leukemia subgroups that require additional leukemogenic events, and therefore, provide the genetic and molecular basis to support the Delayed Infections Hypothesis for leukemia progression in children. Disclosures No relevant conflicts of interest to declare.

Tissue transglutaminase (tTGase) regulates various biological processes, including extracellular matrix organization, cellular differentiation, and apoptosis. Here we report the protective role of tTGase in the cell death that is induced by the tumor necrosis factor alpha (TNF-alpha) and ceramide, a product of the TNF-alpha signaling pathway, in human neuroblastoma SH-SY5Y cells. Treatment with retinoic acid (RA) induced the differentiation of the neuroblastoma cells with the formation of extended neurites. Immunostaining and Western blot analysis showed the tTGase expression by RA treatment. TNF-alpha or C(2) ceramide, a cell permeable ceramide analog, induced cell death in normal cells, but cell death was largely inhibited by the RA treatment. The inhibition of tTGase by the tTGase inhibitors, monodansylcadaverine and cystamine, eliminated the protective role of RA-treatment in the cell death that is caused by TNF-alpha or C(2)-ceramide. In addition, the co-treatment of TNF-alpha and cycloheximide decreased the protein level of tTGase and cell viability in the RA-treated cells, supporting the role of tTGase in the protection of cell death. DNA fragmentation was also induced by the co-treatment of TNF-alpha and cycloheximide. These results suggest that tTGase expressed by RA treatment plays an important role in the protection of cell death caused by TNF-alpha and ceramide.

We have investigated a novel method to monitor real changes of intracellular ROS by the use of CMH2TMRos (a reduced form of MitoTracker orange) in Swiss 3T3 fibroblasts. Arachidonic acid induced a rapid increase of CMTMRos fluorescence with a maximal elevation at 120–150 sec, which was determined by scanning every 10 sec with a confocal microscope. The fluorescence increase by arachidonic acid was completely inhibited by 2-MPG but not by catalase, indicating a major contribution of superoxide to the oxidation of CMH2TMRos. Incubation with glucose oxidase, exogenous H2O2, KO2 and lysophosphatidic acid also increased the CMTMRos fluorescence, which was blocked by 2-MPG. These results suggested that CMH2TMRos is a useful fluorophore for real-time monitoring of intracellular ROS and also indicated that CMH2TMRos detects primarily superoxide in cells even though the fluorophore can be oxidized by both superoxide and H2O2.

Background TCF3-PBX1 is one of the most common recurrent translocations which define distinct subtypes of B-lineage Acute Lymphoblastic Leukemia (B-ALL). Patients with TCF3-PBX1 B-ALL have poor clinical outcome, however, the molecular mechanisms underlying poor outcome are poorly understood. It is critical to identify the cellular processes that contribute to the biological and clinical features of this form of ALL and to develop new targeted therapeutic strategies to improve the outcome. Results We first performed ChIP-seq with antibodies against the fusion oncoprotein TCF3-PBX1 in the human B-ALL cell line 697 and found that the pre–B cell receptor (pre-BCR) genes (IGLL1 or λ5, VpreB, CD79A, CD79B) and µ-chain enhancer regions were directly bound by the fusion protein. Gene expression microarray data showed that the pre-BCR signaling genes (IGLL1, VpreB, IGHM, BLK, LCK, SYK, LYN, SRC and BLNK) were overexpressed in TCF3-PBX1, but not other cytogenetic subtypes of B-ALL (n=132 B-ALL patient samples, St. Jude), suggesting a unique high pre-BCR activity in TCF3-PBX1 ALL. Further flow cytometry analysis using µ-chain specific antibodies showed a strong Ca2+ signal in TCF3-PBX1, but not other subtypes of ALL (n=27). The sequencing analysis on IGHM locus in 148 primary B-ALL samples showed that 100% cases of TCF3-PBX1 (n=8) carried functional IGHM VHDJH gene rearrangements, however only 17% for BCR-ABL1 (n=57), 0% for MLL-AF4 (n=7), 31% for ETV6-RUNX1 (n=13), 3.3% for hyperdiploid (n=30), 20% for sporadic (n=20) and 31% for normal karyotype (n=13) ALL. These data demonstrates that TCF3-PBX1 ALL has an unusual spectrum of high activity of pre-BCR signaling. The transcriptional repressor BCL6 has been identified as a critical survival factor in diffuse large B-cell lymphoma. We found that BCL6 was highly expressed in TCF3-PBX1 as compared to other subtypes of ALL by our Western blot (n=15) and also by gene expression microarray data from three B-ALL clinical trials: COG P9906 (n=207), ECOG E2993 (n=191) and St. Jude ALL (n=132). The clinical data showed that high expression of BCL6 correlates with poor clinical outcome. Those data suggested BCL6 might play a critical oncogene role in TCF3-PBX1 ALL. The TCF3-PBX1 ChIPseq data showed no binding enrichment on BCL6 locus. However, forced expression of pre-BCR components (µ-chain and BLNK) resulted in up-regulation of BCL6 (Western blot), suggesting BCL6 upregulation in TCF3-PBX1 ALL was induced by pre-BCR but not the fusion protein binding. To further test this, we applied SYK and BTK inhibitors to treat TCF-PBX1 ALL cells and found that they dramatically decreased BCL6 mRNA and protein levels and also induced cell apoptosis, suggesting BCL6 might be a therapeutic target for TCF3-PBX1 ALL. To test this, we transduced a primary TCF3-PBX1 B-ALL xenograft sample with a dominant-negative BCL6-mutant (BCL6-DN). Expression of BCL6-DN rapidly induced cell death. When treating primary TCF3-PBX1 ALL cells with the specific BCL6 peptide inhibitor RI-BPI and the small molecule inhibitor PU-H71, they significantly induced cell cycle arrest, compromised colony formation, and prevented leukemia-initiation in transplant recipient mice. Collectively, those data indicates BCL6 is a new therapeutic target for TCF-PBX1 ALL. Dasatinib is a second generation tyrosine kinase inhibitor (TKI) targeting both BCR-ABL1 and Src kinase. We hypothesized that TCF3-PBX1 ALL cells would be more sensitive to Dasatinib than other TKIs because Dasatinib inhibits pre-BCR signaling and hence BCL6. We treated primary TCF3-PBX1 B-ALL cells with Dasatinib and Nilotinib, and found that BCL6 protein expression was abolished with Dasatinib, but no changes with Nilotinib. We also observed a >90% reduction in viability of leukemia cells with 50nM Dasatinib, however only ∼50% cell death at a much higher concentration of Nilotinib (1000nM). These data indicates that Dasatinib can efficiently kill TCF3-PBX1 leukemia cells by inhibiting pre-BCR and BCL6. Conclusions Our study identified TCF3-PBX1 B-ALL as a unique form of ALL which has an unusual high activity of pre-BCR signaling. BCL6 was upregulated by pre-BCR, which we showed was required for proliferation and survival of TCF3-PBX1 ALL cells. Targeting pre-BCR and BCL6 using Dasatinib and specific BCL6 inhibitors (RI-BPI or PU-H71) induced apoptosis of primary TCF3-PBX1 B-ALL cells in vitro and in vivo. Disclosures: No relevant conflicts of interest to declare.

We have investigated possible roles of RhoA and H2O2 in the elevation of intracellular Ca2+ ([Ca2+]i) by phosphatidic acid (PA) in Rat-2 fibroblasts. PA induced a transient elevation of [Ca2+]i in the presence or absence of EGTA. Lysophosphatidic acid (LPA) also increased [Ca2+]i, but the sustained Ca2+response was inhibited by EGTA. LPA stimulated the production of inositol phosphates, but PA did not. In the presence of EGTA, preincubation with LPA completely blocked the subsequent elevation of [Ca2+]i by PA, but not vice versa. PA stimulated the translocation of RhoA to the particulate fraction as did LPA. Scrape loading of C3 transferase inhibited the transient Ca2+ response to PA, but not to LPA, suggesting an essential role of RhoA in the elevation of [Ca2+]i by PA. H2O2 also induced a transient increase of [Ca2+]i as did PA. H2O2 scavengers, catalase andN-acetyl-l-cysteine, completely blocked the rise of [Ca2+]i stimulated by PA, but not by LPA. Furthermore, preincubation with PA blocked the subsequent Ca2+ response to H2O2, and the incubation with H2O2 also blocked the PA-induced rise of [Ca2+]i. Thus, it was suggested that PA stimulated Ca2+ release from PA-sensitive, but not inositol 1,4,5-trisphosphate-sensitive, Ca2+ stores by the activation of RhoA and intracellular H2O2.

Abstract 26 Background: Childhood acute lymphoblastic leukemia (ALL) typically arises from a pre-leukemic pre-B cell clone, which was established in utero (Greaves and Wiemels, 2003). This led to a scenario, in which the initial prenatal lesion is followed by a series of additional transforming events, which ultimately cause malignant transformation of a pre-B cell clone. For instance, the TEL-AML1 gene rearrangement defines the most frequent type of childhood ALL and is detected in ∼1% cord blood samples compared to the cumulative risk for TEL-AML1 ALL at 1:14,000. These findings support the notion that covert pre-leukemic clones are frequent but only a small minority of them develop into frank pre-B leukemia after critical secondary genetic lesions were acquired. The postnatal mechanism(s) that drive the evolution of the fetal pre-leukemic clone towards childhood ALL are not known. Hypothesis: We have recently demonstrated that aberrant somatic hypermutation activity of AID propagates progression of CML into lymphoid blast crisis (Klemm et al., 2009) and clonal evolution of acute lymphoblastic leukemia (Gruber et al., 2010). Here we test the hypothesis that premature expression of AID in human pre-B cells promotes the acquisition of secondary genetic lesions and propagates the clonal evolution of a pre-leukemic pre-B cell towards childhood ALL. Results: We performed a comprehensive analysis of human B cell development in bone marrow samples from two children carrying deleterious mutations of the IL7RA gene encoding one chain of the human IL7 receptor. As opposed to normal human pre-B cells, pre-B cells from IL7RA-mutant patients carried somatically mutated immunoglobulin genes. Premature hypermutation in IL7Rα-deficient pre-B cells was consistent with aberrant expression of AID in these cells. This led to the hypothesis that signaling via IL7Rα suppresses premature activation of AID-dependent hypermutation. To test this hypothesis, we stimulated mouse pre-B cells with LPS in the presence or absence of IL7, which is normally abundantly present in the bone marrow. While pre-B cells did not respond to LPS in the presence of IL7, IL7 withdrawal dramatically sensitized pre-B cells to LPS exposure: in the absence of IL7, LPS-stimulation of pre-B cells resulted in similar AID protein levels as in splenic germinal center B cells, where AID is normally active. We confirmed these observations studying pre-B cells from an AID-GFP reporter transgenic mouse strain. While LPS resulted in ∼2% AID-GFP+ cells in the presence of IL7, the fraction of AID-GFP+ cells increased to ∼45% when IL7 was removed. Since IL7Rα signaling involves Stat5 phosphorylation, we studied inducible deletion of both Stat5a and Stat5b in Stat5-fl/fl pre-B cells. Inducible deletion of Stat5a and Stat5b in pre-B cells had the same effect as IL7 withdrawal and led to transcriptional de-repression of AID. IL7Rα/Stat5 signaling likely involves negative regulation of FoxO3A via AKT since expression of a constitutively active FoxO3A mutant potentiated AID expression in pre-B cells. We next searched for a normal pre-B cell subset, in which loss of IL7Rα/Stat5 signaling occurs naturally. Since inducible activation of pre-B cell receptor signaling results in downregulation of IL7Rα surface expression, we tested pre-B cell receptor-positive stages of B cell development. Interestingly, AID mRNA levels were increased by >10-fold at the transition from IL7Rα-positive Fraction C’ pre-B cells to IL7Rα-negative Fraction D pre-B cells. Conclusion: AID is a tightly controlled mutator enzyme, which diversifies immunoglobulin genes upon antigen-encounter of germinal center B cells. The factors that prevent premature expression of AID in pre-germinal center stage B cells were not known. Here, we here we report a novel, IL7Rα/Stat5-dependent mechanism by which pre-B cells are rendered non-responsive to antigen-dependent upregulation of AID. Attenuation of the IL7Rα/Stat5 signal occurs naturally in Fraction D pre-B cells. As a consequence, Fraction D pre-B cells express significant levels of AID for a short time. We propose that Fraction D pre-B cells represent a subset of increased genetic vulnerability in the natural history of childhood ALL. Enlargement of the Fraction D pool or extension of the time window during which pre-B cells are at the Fraction D stage, may increase the risk to acquire secondary genetic lesions towards the development of childhood ALL. Disclosures: No relevant conflicts of interest to declare.

Abstract 765 BCR-ABL1 tyrosine kinase inhibitors (TKI) are widely used for the treatment of patients with Ph+ ALL and CML. To elucidate mechanisms of TKI-resistance in Ph+ ALL, we studied gene expression changes of a set of 11 primary cases of Ph+ ALL in response to TKI-treatment (16 hours; 2 μmol/l Imatinib). Sorting genes based on the ratio of gene expression values in the presence vs the absence of TKI-treatment, the BCL6 gene consistently ranked #1 in this analysis. As confirmed by quantitative RT-PCR and Western blotting, BCL6 is upregulated by 60- to 90-fold in response to TKI-treatment of Ph+ ALL cells. The BCL6 transcription factor functions as a protooncogene in germinal center (GC)-derived B cell lymphomas. We focused our analysis on BCL6, because it functions a transcriptional repressor of p53 and other cell cycle check point regulators and thereby protects GC-derived B cell lymphoma cells from apoptosis. Of note, BCL6 protein levels in TKI-treated Ph+ ALL cells were as high as in GC-derived B cell lymphoma. We hypothesize that dramatic upregulation of the BCL6 gene in response to TKI-treatment represents a defense mechanism of Ph+ ALL to evade cell death induced by activation of p53 and cell cycle checkpoint regulators. We tested the role of BCL6 in Ph+ ALL in a genetic loss-of-function experiment: To this end, B cell precursors from bone marrow of BCL6+/+ and BCL6−/− mice were transformed with BCR-ABL1. Upon treatment with 1 mmol/l Imatinib for three days, viability of BCL6−/− BCR-ABL1 ALL cells (0.4% ± 0.2%) was lower by two log orders compared to BCL6+/+ leukemia cells (46.0% ± 8.2%; p=0.001). Consistent with our hypothesis that p53 and other cell cycle checkpoint regulators are transcriptional targets of BCL6 in Ph+ ALL, we observed in a comprehensive gene expression analysis that mRNA levels of p53, p21, Arf and p27 were significantly higher in BCL6−/− compared to BCL6+/+ and BCR-ABL1 ALL cells (confirmed by quantitative RT-PCR). To identify target genes of the BCL6 transcriptional repressor, we performed a ChIP-chip analysis for BCL6 both in Ph+ ALL and GC-derived B cell lymphoma. Among the 1,235 target genes of BCL6 in Ph+ ALL, 736 were shared targets with GC-derived B cell lymphoma and 499 were exclusive for Ph+ ALL. These findings suggest that the function of BCL6 in Ph+ ALL does not replicate all aspects of its function in GC-B cell lymphoma. Single locus ChIP analysis confirmed strong recruitment of BCL6 to the promoter regions of the p53, p21 (CDKN1A), Arf (CDKN2A) and p27 (CDKN1B) genes in human Ph+ ALL cells after TKI-treatment. In a genetic loss-of-function experiment, we tested whether transcriptional suppression of p53, p21, Arf and p27 represents a major function of BCL6-upregulation in response to TKI-treatment in Ph+ ALL. To this end, we measured sensitivity of wildtype and Arf−/−, p53−/−, p21−/− and p27−/− BCR-ABL1 ALL cells to either Imatinib alone or to a combination of Imatinib and a novel BCL6 peptide inhibitor (RI-BPI). TKI-treatment alone had approximately the same effect in wildtype BCR-ABL1 ALL cells compared to Arf−/−, p53−/−, p21−/− and p27−/− counterparts. However, when TKI-treatment was combined with BCL6 inhibition (5 μmol/l RI-BPI), the viability of Arf−/−, p53−/−, p21−/− and to lesser degree, p27−/−BCR-ABL1 ALL cells was significantly higher than survival of the respective wildtype control leukemia. These findings confirm that transcriptional suppression of Arf, p53 and p21 (to lesser extent p27) represents an important characteristic of BCL6-mediated drug-resistance in response to TKI-treatment. We next tested the potential therapeutic usefulness of concomitant TKI-treatment and BCL6 peptide inhibition: To this end, 2 × 106 BCR-ABL1 ALL cells were luciferase-labeled and injected into sublethally irradiated NOD/SCID mice. Mice were treated six times between days 7 and 21 after leukemia cell injection with either the BCR-ABL1 kinase inhibitor Nilotinib (75 mg/kg) alone or a combination of Nilotinib and RI-BPI (20 mg/kg). Addition of RI-BPI resulted in significantly prolonged median survival (29 days) compared to Nilotinib alone (22 days; 12 mice per group; p=0.004). We conclude that BCL6-mediated transcriptional repression of p53 and other cell cycle checkpoint regulators represents a novel and critical mechanism of drug-resistance in Ph+ ALL. Combination of TKI-treatment with BCL6 peptide inhibition represents a new promising approach to target drug-resistance in Ph+ ALL. Disclosures: No relevant conflicts of interest to declare.

Abstract 91 Through DNA strand breaks resulting from somatic hypermutation and class-switch recombination, germinal center (GC) B cells are exposed to a high level of DNA damage stress. At the GC stage of development, B cells are protected against apoptosis by specific expression of BCL6, which functions as transcriptional repressor of genes in the DNA damage response pathway. In the absence of BCL6, GC formation is abrogated. During normal B cell development, BCL6 expression was only found in GCs, where the secondary B cell repertoire is shaped. Extensive DNA damage, however, also occurs during the development of the primary B cell repertoire in the bone marrow. B cell precursors in the bone marrow sustain DNA damage during V(D)J recombination at immunoglobulin heavy and light chain loci. It is currently unclear, through which mechanisms early B cell precursors are protected against extensive DNA damage stress caused by V(D)J recombination. Here we report that BCL6 plays a critical role during early B cell development by protecting pre-B cells from DNA damage-induced apoptosis during V(D)J recombination. At the transition from IL7-dependent to IL7-independent stages of B cell development, BCL6 is activated, reaches similar expression levels as in GC B cells. Compared to IL7-dependent pro-B cells and large cycling pre-B cells, BCL6 mRNA and protein levels in IL7-independent small resting pre-B cells were increased by 60- to 90-fold, respectively. We identified STAT5 as a critical negative regulator of BCL6 downstream of IL7 receptor signaling in pre-B cells. Expression of a constitutively active STAT5 mutant prevented BCL6 upregulation in differentiating pre-B cells at the transition from IL7-dependent to IL7-independent stages of B cell development. BCL6 function was then tested in bone marrow precursor cells from BCL6−/− and BCL6+/+ mice: Comparing the gene expression pattern of BCL6−/− and BCL6+/+ pre-B cells, we found that BCL6 is required for transcriptional repression of the ARF (Cdkn2a), p21 (Cdkn1a), Gadd45a and p53 genes, which all contribute to cellular senescence and cell cycle arrest. In agreement with gene expression analyses, ChIP-chip and single-locus q-ChIP studies identified ARF (Cdkn2a), p21 (Cdkn1a), Gadd45a and p53 as transcriptional targets of BCL6 in pre-B cells. BCL6-dependent transcriptional repression of these genes in pre-B cells is critical because BCL6+/+ but not BCL6−/− pre-B cells were capable to proliferate in vitro and to form pre-B cell colonies in semisolid agar. Of note, peptide-inhibition of BCL6 suppressed growth and colony formation in ARF+/+ but not ARF−/− pre-B cells, suggesting that ARF-deficiency rescues lack of BCL6 function. We conclude that BCL6-mediated transcriptional repression of ARF is critical for pre-B cell self-renewal. To determine whether BCL6 function is also required for normal early B cell differentiation in vivo, we performed a comprehensive analysis of B cell differentiation stages in bone marrow from BCL6−/− and BCL6+/+ mice. In agreement with previous studies, the overall number of B cell precursors in the bone marrow was only slightly reduced and pro-B cell and large pre-B cell populations were normal. However, the pools of small-resting pre-B cells and new emigrant B cells were reduced in BCL6−/− mice by 3- and 7-fold, respectively. While the overall numbers of mature B cells in BCL6−/− mice were normal, we found that their clonal repertoire was extremely restricted. Using spectratype analysis, we found a broad polyclonal primary B cell repertoire in BCL6+/+ mice, whereas the B cell repertoire in their BCL6−/− counterparts was strictly oligoclonal. We conclude that pre-B cell self-renewal and polyclonal B cell production critically depends on BCL6. While the self-renewal defect of BCL6-deficient pre-B cells can be numerically compensated by increased proliferation at later stages of development, the diversity of the B cell repertoire in BCL6−/− mice is permanently restricted. We conclude that BCL6 is required for pre-B cell self-renewal and the formation of a normal polyclonal B cell repertoire. Disclosures: No relevant conflicts of interest to declare.

Abstract 2167 Poster Board II-144 The transcriptional repressor BCL6 was discovered as a potent protooncogene in diffuse large B-cell lymphoma (DLBCL) and is also expressed in normal germinal center (GC) B cells. BCL6 protects DLBCL and GC B cells against DNA damage-induced apoptosis by suppressing target genes including p53, p21, ATR and p27. In preliminary experiments for this study, we found that treatment of BCR-ABL1-driven Ph+ ALL cells with Imatinib results in a 60- to 90-fold up-regulation of BCL6, reaching protein levels as high as in DLBCL and GC B cells. Since the oncogenic BCR-ABL1 kinase also drives leukemic transformation in CML cells, we studied regulation of BCL6 in human CML cells upon TKI-treatment. Like in Ph+ ALL, TKI-treatment resulted in rapid BCL6 mRNA and protein upregulation in CML cells, albeit to lesser degree than in Ph+ ALL and DLBCL. We hypothesized that upregulation of BCL6 in CML cells in response to TKI-treatment serves the same function as in DLBCL and protects from p53-mediated apoptosis. Through transcriptional repression of p53, p21, ATR and p27 as in DLBCL, upregulation of BCL6 in response to TKI-treatment would represent a defense mechanism of CML cells to evade TKI-induced cell death. We tested the role of BCL6 in CML in a genetic loss-of-function experiment: To this end, myeloid progenitor cells from bone marrow of BCL6+/+ and BCL6-/- mice were transformed with BCR-ABL1. Compared to their BCL6+/+ counterparts, BCL6-/- mouse CML cells were highly sensitive to Imatinib-treatment. In a complementary approach, we treated human CML cells with either Imatinib alone or with a novel BCL6 peptide inhibitor (Retro-inverso BCL6-peptide inhibitor, RI-BPI). Consistent with findings in BCL6-/- mouse CML cells, also human CML cells were significantly sensitized to Imatinib by BCL6 inhibition (using 5 mmol/l RI-BPI). Of note, the BCL6 inhibitory peptide RI-BPI alone induced no significant toxicity in human CML cells but synergized with Imatinib-treatment. Since RI-BPI alone had no cytotoxic effect on CML cells, we tested whether loss of BCL6 function affects self-renewal capacity of CML cells. This possibility was explored in two complementary colony formation assays: In one set of experiments, we tested the ability of BCL6+/+ and BCL6-/- mouse CML cells to form colonies in semisolid methylcellulose agar. The comparison between BCL6+/+ (94 ± 11 colonies) and BCL6-/- ( Disclosures: No relevant conflicts of interest to declare.

SummaryStudying 830 pre-B ALL cases from four clinical trials, we found that human ALL can be divided into two fundamentally distinct subtypes based on pre-BCR function. While absent in the majority of ALL cases, tonic pre-BCR signaling was found in 112 cases (13.5%). In these cases, tonic pre-BCR signaling induced activation of BCL6, which in turn increased pre-BCR signaling output at the transcriptional level. Interestingly, inhibition of pre-BCR-related tyrosine kinases reduced constitutive BCL6 expression and selectively killed patient-derived pre-BCR+ ALL cells. These findings identify a genetically and phenotypically distinct subset of human ALL that critically depends on tonic pre-BCR signaling. In vivo treatment studies suggested that pre-BCR tyrosine kinase inhibitors are useful for the treatment of patients with pre-BCR+ ALL.

Background Toll-like receptor 2 (TLR2) plays a critical role in mediating inflammatory/immune responses against bacterial pathogens in lung. Streptococcus pneumoniae (S. pneumoniae) and nontypeable Haemophilus influenzae (NTHi) were previously reported to synergize with each other to induce inflammatory responses. Despite the relatively known intracellular signaling pathways involved in the synergistic induction of inflammation, it is still unclear if both bacterial pathogens also synergistically induce expression of surface TLR2. Results Here we provide direct evidence that S. pneumoniae synergizes with NTHi to upregulate TLR2 expression in lung and middle ear of the mice. Pneumolysin (PLY) appears to be the major virulence factor involved in this synergism. Moreover, S. pneumoniae PLY induces TLR2 expression via a TLR4-MyD88-NF-κB-dependent signaling pathway. Interestingly, tumor suppressor CYLD acts as a negative regulator of S. pneumoniae-induced TLR2 up-regulation via negative-crosstalk with NF-κB signaling. Conclusion Our study thus provides novel insights into the regulation of TLR2 expression in mixed bacterial infections.