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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.

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 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.