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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.

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.

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.

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.

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.

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.

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 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.