ALBERT

Description: Asparaginase (ASNase) is one of the cornerstones of the multi-drug treatment protocol that is used to treat acute lymphoblastic leukemia (ALL) in pediatric and adult patients. Despite the fact that ASNase has been used in ALL treatment protocols for decades, little is known about the biodistribution and the mechanism of ASNase turnover in vivo. A large inter-individual variation in ASNase pharmacokinetics is observed in patients. While elevated ASNase levels are associated with an increase in adverse events, underexposure, frequently caused by antibody mediated clearance, seriously reduces therapeutic efficacy. To date, it is not possible to predict pharmacokinetics of ASNase in individual patients and therefore current therapeutic protocols are supported by frequent monitoring of ASNase levels and adjustments of administration schemes. We used an in vivo imaging approach to study ASNase biodistribution and pharmacodynamics in a mouse model and provide in vitro and in vivo evidence that identifies the endo-lysosomal protease Cathepsin B in macrophages as a critical component of ASNase degradation. Results/Discussion Mice were injected with 111Indium-labeled ASNase and biodistribution was monitored by quantitative microSPECT/CT scans and ex vivo analysis of organs using a gamma counter. Over time, ASNase accumulated in the liver and particularly the spleen and the bone marrow. We hypothesized that macrophages in these organs, efficiently take up the ASNase, thereby rapidly clearing the active enzyme from the blood. Immunohistochemical analysis confirmed the presence of ASNase in cells positive for the murine macrophage marker F4/80. To confirm the importance of macrophage populations in ASNase clearance, we depleted mice from phagocytic cells by injection of clodronate liposomes, and studied ASNase biodistribution and kinetics. Indeed, clodronate pretreatment significantly diminished the accumulation of ASNase in the liver, spleen and the bone marrow while doubling the circulatory half-life of serum ASNase activity. We conclude from these experiments that macrophages determine the pharmacokinetics of asparaginase, which raises the question whether rapid clearance of the drug by bone marrow resident macrophages will negatively affect the depletion of asparagine in the bone marrow niche. We previously linked a germline mutation in the gene encoding endosomal protease Cathepsin B to strongly diminished asparaginase degradation in a pediatric ALL patient. To connect the macrophage mediated clearance to the proposed role of Cathepsin B in ASNase degradation, we studied the contribution of this protease in macrophage-mediated degradation of asparaginase. We used cell lines to show that Cathepsin B expression is induced during differentiation from monocytes towards macrophages. This is consistent with our finding that macrophages, but not monocytes, are capable of degrading ASNase. Furthermore, we used both chemical inhibition and RNAi mediated knockdown of Cathepsin B to show that this protease is required for ASNase degradation in these macrophages. Finally, by comparing Cathepsin B knockout mice with wildtype littermates, we demonstrated that loss of Cathepsin B activity significantly delayed clearance of serum asparaginase, consistent with a prominent role for this lysosomal protease in ASNase turnover. In conclusion, by using in vivo imaging we showed that asparaginase is efficiently cleared from the circulation by macrophages. In particular, bone marrow resident macrophages may provide a protective environment for leukemic cells by effectively removing the therapeutic protein from the bone marrow niche. However, both the prominent role of macrophages and the importance of the lysosomal protease Cathepsin B in asparaginase clearance, may allow the rational design of a next generation asparaginase. Disclosures Metselaar: Enceladus Pharmaceuticals: Employment, Equity Ownership.

Description: Background Glucocorticoids (GCs) such as prednisolone and dexamethasone are critical components of multi-agent chemotherapy regimens used in the treatment of acute lymphoblastic leukemia (ALL). Children with ALL are stratified into risk groups based on diagnostic features (i.e. age and cytogenetics) and therapy response. It has been established that the initial response to prednisolone is a major prognostic factor. Moreover, at relapse, de novo or acquired resistance to GCs is common and represents an important determinant in treatment failure. Recent studies performed by us and others have identified IKZF1 gene deletions and mutations as an independent prognostic factor that predicts prognosis and treatment outcome of children with B cell precursor ALL (BCP-ALL). These monoallelic IKZF1 gene deletions either affect the whole gene or may result in expression of dominant-negative IKZF1 isoforms due to intragenic deletions. However, it has not been established whether loss of IKZF1 function directly impacts the response to glucocorticoids. Results We examined whether haplodeficiency for Ikzf1 gene expression in mouse lymphocytes affects glucocorticoid-induced apoptosis. Splenocytes from Ikzf1+/- knockout mice were activated with lipopolysaccharide (LPS) and treated with increasing concentrations of either prednisolone or dexamethasone for 48 hours. B-lymphocytes haplodeficient for IKZF1 showed a significantly enhanced survival after treatment with GCs compared to wild type cells, as measured in an MTS assay and by AnnexinV staining. In case of prednisolone, the inhibitory concentration (IC50) was about ∼200-fold higher in the Ikzf1+/- splenocytes as compared to the wild-type cells. Gene expression analysis revealed that Ikzf1+/- splenocytes displayed lower overall expression levels as well as diminished transcriptional activation of several glucocorticoid receptor (GR)-induced target genes (i.e. Sgk1, Irs2, Zfp36L2). Furthermore, in luciferase reporter assays we established that IKZF1 overexpression enhances GR-mediated transcriptional activation in response to prednisolone. Finally, lentivirus-mediated IKZF1-shRNA expression in Nalm6 cell line, which reduces endogenous IKZF1 protein levels to around 50%, inhibits prednisolone and dexamethasone-induced apoptosis, demonstrating that also in human leukemia cells reduced IKZF1 expression levels protect against GC-induced cell death. In conclusion, our data provide evidence that loss of IKZF1 function mediates resistance to glucocorticoid-induced apoptosis, which may contribute to the poor outcome of IKZF1-deleted BCP-ALL. Disclosures: No relevant conflicts of interest to declare.

Description: Resistance to glucocorticoids (GCs) is a major clinical problem in the treatment of acute lymphoblastic leukemia (ALL), but the underlying mechanisms are not well understood. Although mutations in the glucocorticoid receptor (GR) gene can give rise to therapy resistance in vitro, acquired somatic mutations in the GR are rarely encountered in patients. Here we report that the protein encoded by the BTG1 gene, which is frequently deleted in (pediatric) ALL, is a key determinant of GC responsiveness. Using RNA interference, we show that loss of BTG1 expression causes GC resistance both by decimating GR expression and by controlling GR-mediated transcription. Conversely, reexpression of BTG1 restores GC sensitivity by potentiating GC-induced GR expression, a phenomenon known as GR autoinduction. In addition, the arginine methyltransferase PRMT1, a BTG1-binding partner and transcriptional coactivator, is recruited to the GR gene promoter in a BTG1-dependent manner. These results implicate the BTG1/PRMT1 complex in GR-mediated gene expression and reveal that deregulation of a nuclear receptor coactivator complex can give rise to GC resistance. Further characterization of this complex as part of the GR regulatory circuitry could offer novel opportunities for improving the efficacy of GC-based therapies in ALL and other hematologic malignancies.

Description: Background Treatment outcome in acute lymphoblastic leukemia (ALL) has improved over the past 30 years, with overall survival rates of ∼45% in adults and ∼85% in children. Gross cytogenetic abnormalities, including numerical changes and chromosomal translocations, are of considerable prognostic value in both pediatric and adult ALL. In addition, we and others have recently identified novel molecular markers associated with a poor outcome in ALL, including deletions of the lymphoid transcription factor IKZF1. In order to identify downstream signaling events associated with these genetic alterations, we performed an integrated analysis of genomic abnormalities, including copy number alterations, sequence mutations and chromosomal translocations, with alterations in protein expression and modification. Methods A cohort of 91 precursor B-ALL cases treated at M.D. Anderson Cancer Center in Houston, USA, including 82 newly diagnosed cases and 5 diagnosis-relapse pairs was used for this study. The cohort consisted of 6 children (age 1-6), 30 young adults (age 15-39) and 45 adults (age〉39), and 20 patients carried a BCR-ABL1 chromosomal translocation. Copy number alterations in eight genes frequently deleted in ALL (IKZF1, PAX5, EBF1, RB1, CRLF2, CDKN2A/2B, BTG1, and ETV6) were determined by multiplex ligation-dependent probe amplification analysis. IKZF1 deletions were associated with relapse (Pearson's chi-square test, p=0.009), and the presence of BCR-ABL1 translocation (p=0.032). Protein expression and modification levels were determined by probing Reverse Phase Protein Arrays (RPPA) containing protein lysates of all above samples with 128 rigorously validated antibodies including 34 phospho-specific antibodies. Hierarchical clustering analysis was used to determine which (phospho)proteins are differently expressed in genetic subsets of ALL. The significance of correlations was determined using two-sample t-test, with correction for multiple testing (Beta-Uniform Mixture model). Results We identified clustering of cases with a BCR-ABL1 chromosomal translocation (p=0.01; false discovery rate (FDR)=0.1), IKZF1-deletions (p=0.01, FDR=0.072), RB1-deletions (p=0.03, FDR=0.43) and EBF1 deletions (p=0.05, FDR=0.63). As expected RB1 deletion positive cases were characterized by decreased levels of (phospho)-RB1 and increased levels of cyclin E, illustrating the validity of our approach. EBF1-deleted cases showed relatively high levels of SHIP1, SSBP2 and phospho-STAT5, and lower levels of FAK and LYN. The protein signatures of BCR-ABL1-positive cases and IKZF1-deletion positive cases largely overlapped, and were characterized by elevated levels of (phospho)PKCα, SMAD1, phospho-STAT3, and phospho-STAT5 and lower levels of LYN and cyclinD3 (Figure 1). In total 70% of the BCR-ABL1-positive cases carried an IKZF1 deletion and several BCR-ABL1-negative cases with similar RPPA signature could be identified, all of which were IKZF1-deletion positive. These cases may represent the “BCR-ABL1-like” cases that were previously identified using gene expression signatures (Mullighan et al. 2009, NEJM 360:470-480; Den Boer et al. 2009, Lancet Oncol. 10:125-134), and could reflect activation of cAbl or other cellular tyrosine kinases. Together, we conclude that integrated analysis of genetic and proteomic aberrations identified protein signatures downstream of recurrent mutational events in ALL, a strategy that promises to facilitate the discovery of novel therapeutic targets in ALL and may aid in the identification of (high risk) patients that would benefit from tyrosine kinase inhibition. Disclosures: No relevant conflicts of interest to declare.

Description: We and others have shown that the B cell Translocation Gene 1 (BTG1) locus is affected by genomic deletions in 9% of pediatric acute lymphoblastic leukemia (ALL) patients. The fact that multiple subclones carrying distinct deletions can be present in individual patients suggests that interfering with normal BTG1 function provides a selective growth advantage to leukemic cells. However, it remains unclear how loss of BTG1 promotes clonal outgrowth. We detected an up to 15-fold increases of BTG1 expression when lymphoid cells were exposed to various challenge conditions, including nutrient limitation and ER stress induction. To test for a functional role for BTG1 in the cellular response to stress, we cultured BTG1 knockout cells in medium without glucose or amino acid (Figure 1) and found that BTG1 knockout cells show a 20-30% improved survival rate as compared to wildtype cells.Figure 1BTG1 knockout cells are resistant to Asparaginase treatment.Figure 1. BTG1 knockout cells are resistant to Asparaginase treatment. As Activating Transcription Factor 4 (ATF4) is a master regulator of cellular stress signaling, we hypothesized that the improved survival after BTG1 loss is regulated via ATF4. By immunoprecipitation experiments, we showed that BTG1 complexes with ATF4. In addition, co-expression of BTG1 attenuates ATF4 transcriptional activity on target gene promoters and suppresses both recombinant and endogenous ATF4 function in these promoter reporter assays (Figure 2).Figure 2BTG1 attenuates ATF4 transcriptional activity.Figure 2. BTG1 attenuates ATF4 transcriptional activity. Although BTG1 possesses no catalytic activity, it functions as a transcriptional co-regulator that acts by recruiting Protein Arginine Methyl Transferase 1 (PRMT1) to transcription factor complexes. By in vitro methylation assays with purified proteins we showed that ATF4 is directly methylated by PRMT1 on a single arginine residue. In addition we found that the PRMT1 interacting domain in BTG1, while dispensable for the BTG1-ATF4 interaction, is essential for the BTG1 mediated suppression of ATF4 function. In search for additional evidence for the functional interaction between BTG1 and ATF4 we performed global expression analysis on murine cells expressing the B cell marker B220. This revealed a significant deregulation of ATF4 target genes in BTG1 knockout cells when compared to wildtype cells. Together, our data indicate that BTG1 suppresses activation of ATF4 in response to cellular stress. Loss of BTG1 function, as it occurs during leukemia development, enhances ATF4 activity, thereby promoting cell survival under cellular stress conditions such as nutrient deprivation or ER stress. Leukemic cells carrying BTG1 deletions may thus benefit from this increased resistance to cellular stress, not only during leukemia development but also during treatment. Hence, targeting the ATF4 stress response pathway may prevent relapse of therapy-resistant leukemic clones. Cells were treated with 2 IU/L Asparaginase for 24 hours. After treatment, cell viability was measured using an MTT assay. The average of 4 independent experiments is plotted with error bars representing the standard error of the mean. A luciferase reporter gene controlled by the ATF4 responsive ASNS promoter region was transfected into HEK293 cells. Asparaginase treatment induces endogenous ATF4 expression, which results in an increase in luciferase signal (Mock transfected cells). Co-expression of BTG1 represses both endogenous ATF4 activity as well as ectopically expressed ATF4 activity as detected by a decrease in luciferase signal. The average of 2 independent experiments is plotted with error bars representing the standard deviation. Disclosures: No relevant conflicts of interest to declare.

Description: Background The chromosomal translocation BCR-ABL1 is frequently present in adult B cell precursor acute lymphoblastic leukemia (BCP-ALL) in about 30% of the patients, while in pediatric BCP-ALL it occurs only in 3% of the patients. However, in both cases the disease is characterized by the almost obligatory presence of IKZF1 gene deletions and mutations, arguing that loss of IKZF1 function is required for oncogenic transformation by p190BCR-ABL1. The IKZF1 gene encodes a transcription factor that belongs to the Ikaros family of zinc-finger proteins, which mainly acts as a transcriptional repressor protein through the recruitment of both HDAC-dependent and HDAC-independent co-repressor molecules. However, in some cases IKZF1 has also been shown to transcriptional activate specific target genes through association with the SWI/SNF chromatin remodeling complexes. We hypothesized that IKZF1-mediated transcription in a direct or indirect manner is modulated by BCR-ABL1 signaling. Therefore, we performed cell biological assays and proteomic studies to investigate the effect of p190BCR-ABL1 expression on IKZF1 protein function. Results Using a luciferase reporter assay employing the human BAX- promoter, we established that IKZF1-induced transcriptional repression was alleviated by p190BCR-ABL1 expression. This effect could be reversed by Imatinib treatment, suggesting that BCR-ABL1 signaling interferes with the normal function of IKZF1. Next, we assessed the effect of p190BCR-ABL1 on doxycycline-induced expression of IKZF1 using the murine lymphoid Tet-on Ba/F3 (TonB) cell line. Gene expression analysis showed that several target genes that are repressed by IKZF1 in TonB cells, such as p16Ink4a, Cnot6, Dscc1 and Tspan5, are transcriptionally induced by co-expression of p190BCR-ABL1. In order to understand how p190BCR-ABL1 signaling affects IKZF1 protein function, mass spectrometry was performed on FLAG-affinity purified IKZF1 from transiently transfected HEK293 cells in the absence or presence of p190BCR-ABL1. These analyses revealed that p190BCR-ABL1 expression induces phosphorylation of IKZF1 on specific serine, threonine and tyrosine residues as well lysine acetylation. Transient transfection of lysine acetyltransferase PCAF (KAT2B) confirmed that IKZF1 is modified by lysine acetylation. Western blot analysis using phospho-specific antibodies showed that IKZF1 is subject to tyrosine phosphorylation by p190BCR-ABL1, both in HEK293 cells and TonB cells. Using an in vitro kinase assay, we demonstrated that IKZF1 can be directly phosphorylated by active recombinant ABL kinase. Conclusion Our studies show that p190BCR-ABL1 signaling induces a multitude of different post-translational modifications on IKZF1, which could modify its properties as transcriptional regulator. We propose that modulation of IKZF1 tumor suppressor function by p190BCR-ABL1 signaling is the driving force for IKZF1 gene deletions in BCP-ALL patients harboring a BCR-ABL1 translocation. Disclosures: No relevant conflicts of interest to declare.

Description: B cell precursor acute lymphoblastic leukemia (BCP-ALL) is one of the most common malignancies in children. In the period 1991-2013, the Dutch Childhood Oncology Group (DCOG) has completed three treatment trials for childhood ALL: ALL8, 9 and 10, each protocol with stratifications into risk-groups (details: www.skion.nl). Although the cure rates increased in these subsequent trials, relapses still occurred in a significant number of children. Since consecutive upfront treatment protocols usually change at multiple levels, genomic alterations that are associated with relapse may also be variable, which could provide insight into the biology underlying therapy failure and relapse. In this study, we characterized the genetic architecture of relapsed BCP-ALL patients within the context of these three Dutch upfront protocols. We identified 3 patient groups based on upfront treatment as follows: Group-1: patients treated upfront with high-amounts of corticosteroids (CS) and relatively mild additional chemotherapy (ALL9 NHR/HR); Group-2: patients treated with high-amounts of CS and intensive additional chemotherapy (ALL10 MR); Group-3: patients treated with low-amounts of CS and moderately-intensive additional chemotherapy (ALL8 SR/MR, ALL10 SR). The number of high-risk patients that relapsed after ALL8 HR and ALL10 HR chemotherapy courses was too low to be included for analysis. We determined, at relapse, the presence of copy number alterations and sequence mutations in 21 recurrently affected genes involved in B-cell development, cell cycle regulation and RAS signaling, in 123 patients that relapsed after treatment in group-1 (n=56), group-2 (n=20) and group-3 (n=47). The number of CREBBP mutations in patients that relapsed after treatment according to group-1 (ALL9) was significantly lower compared to the other two groups, whereas B-cell development alterations were most common in patients that relapsed after treatment according to group-1, mainly due to a higher number of IKZF1 alterations (Figure 1). The high number of relapsed patients with leukemic clones carrying IKZF1 alterations in patients treated with high-amounts of CS and relatively mild additional chemotherapy is in line with our recent finding that IKZF1 is a key determinant of GC-induced apoptosis in normal and leukemic B-cells, and that loss of IKZF1 function confers resistance to dexamethasone, the major treatment component in group-1 (Marke et al., submitted). Additionally, in the group-2 patients treated with high-amounts of CS and highly intensive additional chemotherapy, a lower percentage IKZF1-deleted clones was detected at relapse, indicating that more GC-resistant, IKZF1-deleted clones are killed by the intense chemotherapy given in addition to CS in group-2 patients. Similarly, in the group-3 patients relapsing after treatment with lower amounts of CS and moderately-intensive additional chemotherapy, the percentage of surviving IKZF1-deleted clones was lower than in patients treated with high-amounts of CS. Taken together, our data indicate that the genetic architecture of relapsed BCP-ALL patients depends on the upfront treatment and, in addition, that the poor-prognostic feature of IKZF1-deletions may be more prominent in upfront treatment with high-amounts of CS and relatively mild additional chemotherapy. Figure 1. The frequency of genetic alterations in studied genes in patients that relapsed after treatment according to group-1, 2 and 3. Genes were grouped by their corresponding pathways. Group-1: patients treated upfront with high-amounts of CS and relatively mild additional chemotherapy (ALL9 NHR/HR); Group-2: patients treated with high-amounts of CS and intensive additional chemotherapy (ALL10 MR); Group-3: patients treated with low-amounts of CS and moderately-intensive additional chemotherapy (ALL8 SR/MR, ALL10 SR). Asterisk showed significant difference between upfront treatment groups, **p

Description: Abstract 2458 The bacterially derived enzyme L-Asparaginase (ASNase) is a key component in the multidrug therapy regimens used worldwide to treat pediatric and adult patients with acute lymphoblastic leukemia (ALL), however little is known about the molecular mechanisms that control the pharmacokinetics of this therapeutic protein. As a result, many patients who receive a standardized dose either exceed or do not reach the desired serum concentration. While elevated serum levels are associated with an increase in treatment related morbidity, underexposure seriously compromises therapeutic benefits. In search of molecular factors that determine ASNase turnover in vivo, we investigated a patient with strongly aberrant clearance kinetics. This 3-year old female diagnosed with common ALL suffered from severe ASNase-induced adverse events upon treatment with ErwiniaSNase as a result of strongly elevated serum ASNase levels. Pharmacokinetics data showed a severely delayed ASNase clearance. As a result, serum ASNase levels accumulated to intolerable levels upon repeated administration of the drug. We isolated DNA from peripheral blood mononuclear cells and buccal cells of this patient and performed targeted sequencing on genes suggested to be involved in ASNase clearance. We identified a novel heterozygous mutation in the gene encoding Cathepsin B in the germline of this patient. The mutant allele shows a deletion of a single codon, leading to a deletion of a lysine residue in the C terminus of the protein. We generated an EBV LCL cell line from this patients which showed a 75% reduction in Cathepsin B activity, relative to controls, indicating that this heterozygous mutation has a profound effect on the total Cathepsin B activity. Cathepsin B is normally synthesized as a 37 kD pre-pro enzyme and is processed in a two step process into a mature 2-chain active form. During this process, the protein is transported to the lysosome where it exerts its primary function. Using a combination of biochemical and imaging experiments we show that the mutant Cathepsin B cannot be processed into the mature form and is retained in the endoplasmatic reticulum. ASNase degradation assays demonstrate that this mutant form of Cathepsin B shows a diminished protease activity towards both E.coli and Erwinia ASNase, consistent with the reduced clearance observed in our patient. Cathepsin B and other cellular proteases are either actively secreted or released into the serum as a result of cell lysis. Although we find a variable low but detectable activity of Cathepin B in serum samples, all tested preparations of ASNase were stable upon prolonged incubation in serum, suggesting that serum components are not contributing to ASNase clearance in vivo. Hence, we propose that cellular uptake and subsequent proteolytic degradation of ASNase is the primary mechanism of clearance. In conclusion, we have identified a mutation in protease Cathepsin B and provide evidence that this mutation results in a loss of protease function towards ASNase, which can explain the strongly delayed clearance of ASNase in the patient. Our data suggest that differences in Cathepsin B activity may contribute to the large inter-patient variability in ASNase pharmacokinetics. Furthermore, given the role of Cathepsin proteases in antigen presentation, Cathepsin B may not only provide a target for predicting or controlling ASNase clearance kinetics but inhibition of Cathepsin may also prevent or delay the formation of inhibitory antibodies. Disclosures: Boos: European Erwinase Providers (EUSAPharm): Speakers Bureau; Medac: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Lanvers-Kaminsky:Medac: Speakers Bureau.

Description: In B cell neoplasia, many transcription factors known to be involved in B cell differentiation and commitment, like E2A, EBF1 and PAX5, are frequently targeted by focal deletions, mutations or chromosomal aberrations. Recent studies have shown that the human genes BTG1 and BTG2 are commonly affected by gene alterations in different B cell malignancies, but their role in normal B cell development has not been established. BTG1 and BTG2 can act as transcriptional cofactors through recruitment of the protein arginine N-methyltransferase PRMT1, which mediates arginine methylation of transcription factors, like RUNX1, and on histone 4 arginine 3 (H4R3). Here we report that Btg1 and Btg2 display unique and overlapping functions during mouse B cell development. We observed a reduction in the fraction of B220+ progenitor cells in the bone marrow compartment of the different knockout animals, ranging from a 10% decrease in the Btg2-/-, 20% in Btg1-/- , and 40% in the Btg1-/-;Btg2-/- mice relative to wild-type controls. Deficiency for Btg1, but not Btg2, resulted in reduced outgrowth of IL-7 dependent lymphoid progenitors in methylcellulose, which correlated with a higher fraction of apoptotic cells. Btg2-/- mice showed impaired differentiation at the pre-pro-B, pro-B and pre-B cell stage, while Btg1-deficiency mainly affected later stages of B cell differentiation with reduced numbers of immature B cells. Btg1-/-;Btg2-/- mice displayed additive effects with more significant reduction of B220+ cells predominantly at the pre-B and immature B cell stage. Expression analysis revealed no reduction in the mRNA levels of master regulators E2a, Foxo1, Ebf1 and Pax5 in the absence of Btg1 and Btg2. However, higher expression levels of T cell-specific genes were observed in Btg1-/-;Btg2-/- progenitor B cells, e.g. Cd4, Ikzf2 and Tcf7 (Figure 1), some of which are known to be transcriptional repressed by Ebf1, such as Id2, Gata3, Dtx3l and Notch1. Flow cytometric analyses confirmed increased expression of CD3, CD4 and CD8 markers on CD19+ bone marrow cells lacking Btg1 and Btg2 function. Additionally, we detected enhanced levels of DC, NK and myeloid markers on Btg1-/-;Btg2-/- CD19+ BM cells, indicating that Btg1 and Btg2 repress alternative cell fates during B cell lineage specification, and are required for the maintenance of B cell identity. Biochemical studies showed evidence for a physical association between Ebf1, Btg1/Btg2 and PRMT1. We propose a model in which Btg1 and Btg2 affect the function of the critical B cell transcription factor Ebf1 by recruitment of PRMT1. Figure 1. Aberrant T-lineage expression in progenitor B cells deficient for Btg1 and Btg2. Relative expression levels of Cd4, Runx1, Ikzf2, Tcf7, Id2, Gata3, Notch1 and Dtx3l were determined on cDNA generated from B220+ BM cells of wild-type (WT), Btg1-/-, Btg2-/- and Btg1-/-;Btg2-/- mice by quantitative real-time PCR and normalized to the expression of the housekeeping gene TATA box binding protein (TBP). Data represent the mean and SEM of three independent experiments containing cDNA derived from 2 different biological samples. *, P〈 0.05, **, P〈 0.01, ***, P〈 0.001. Figure 1. Aberrant T-lineage expression in progenitor B cells deficient for Btg1 and Btg2. Relative expression levels of Cd4, Runx1, Ikzf2, Tcf7, Id2, Gata3, Notch1 and Dtx3l were determined on cDNA generated from B220+ BM cells of wild-type (WT), Btg1-/-, Btg2-/- and Btg1-/-;Btg2-/- mice by quantitative real-time PCR and normalized to the expression of the housekeeping gene TATA box binding protein (TBP). Data represent the mean and SEM of three independent experiments containing cDNA derived from 2 different biological samples. *, P〈 0.05, **, P〈 0.01, ***, P〈 0.001. Disclosures No relevant conflicts of interest to declare.

Description: Translocation t(12;21) (p13;q22), giving rise to the ETV6-RUNX1 fusion gene, is the most common genetic abnormality in childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL). The ETV6-RUNX1 translocation arises in utero, but its expression is insufficient to induce leukemia and requires other cooperating genetic lesions for BCP-ALL to develop. Deletions affecting the transcriptional coregulator BTG1 are commonly observed in BCP-ALL (9%), but are significantly enriched in ETV6-RUNX1-positive leukemia (25%). The BTG1 protein displays no intrinsic enzymatic activity but may act by recruiting effector molecules like protein arginine methyltransferase 1 (PRMT1) to specific transcription factors. Here, we show that ETV6-RUNX1 interacts both with BTG1 and PRMT1, and this interaction is lost in c-Kit+Ter-119-Btg1-/- fetal liver (FL) derived hematopoietic progenitors (HPCs). Moreover, targeted deletion of Btg1 enhanced the proliferative capacity of ETV6-RUNX1 in FL-HPCs as measured by enhanced colony-forming and serial replating capacity (Figure 1). The combined loss of Btg1 function and ETV6-RUNX1 expression correlated with strong upregulation of the proto-oncogene Bcl6 and downregulation of BCL6 target genes, such as p19Arf and Tp53 (Figure 2). Similarly, ectopic expression of BCL6 promoted both proliferation and replating capacity of FL-derived progenitor cells in the presence of SCF, FLT3L and IL-7. This phenotype correlated with a fivefold suppression of p19Arf and a twofold suppression of Tp53 expression. Inhibition of BCL6 in a variety of human BCP-ALL cell lines by the peptide inhibitor RI-BPI resulted in decreased proliferation and induction of apoptosis as measured by Annexin-V staining. These included the ETV6-RUNX1-positive cell lines UOC-B6, AT-2 and REH, the BCR-ABL1-positive cell line SD1, as well as Nalm6. Together our results point to a novel role for BCL6 in promoting cell proliferation of primitive progenitor B cells and suggest that targeted inhibition of BCL6 may be effective in the treatment of various BCP-ALL subtypes. Figure 1. Btg1-deficiency enhances the proliferative capacity of early FL-HPCs expressing ETV6-RUNX1. FL-derived hematopoietic progenitor cells (FL-HPCs) (cKit+Ter119-) were isolated from wild-type and Btg1-/- embryos at day 13.5dpc and transduced with control and ETV6-RUNX1 virus. Control and ETV6-RUNX1 transduced FL-HPCs (1x104 cells) were added 48 hours after transduction in B cell specific methylcellulose in the presence of FLT-3L, IL-7 and SCF. Serial replating was performed under identical conditions. Mean colony counts (and SEM) were determined (〉30 cells/colony) after 6-10 days of culture. Data is a representative of 2 independent experiments. *, P〈 0.05, **, P〈 0.01. Figure 1. Btg1-deficiency enhances the proliferative capacity of early FL-HPCs expressing ETV6-RUNX1. FL-derived hematopoietic progenitor cells (FL-HPCs) (cKit+Ter119-) were isolated from wild-type and Btg1-/- embryos at day 13.5dpc and transduced with control and ETV6-RUNX1 virus. Control and ETV6-RUNX1 transduced FL-HPCs (1x104 cells) were added 48 hours after transduction in B cell specific methylcellulose in the presence of FLT-3L, IL-7 and SCF. Serial replating was performed under identical conditions. Mean colony counts (and SEM) were determined (〉30 cells/colony) after 6-10 days of culture. Data is a representative of 2 independent experiments. *, P〈 0.05, **, P〈 0.01. Figure 2. Targeted deletion of Btg1 cooperates with ETV6-RUNX1 in regulating critical effector pathways implicated in leukemia. Relative expression levels of Bcl6, Tp53 and p19arf in empty control (Ctrl) and ETV6-RUNX1 transduced wild-type and Btg1-deficient fetal liver-derived hematopoietic progenitor cells by real-time PCR and normalized to the expression of the housekeeping gene TATA box binding protein (TBP). Data represent the mean and SEM of three independent experiments. *, P〈 0.05, **, P〈 0.01, ***, P〈 0.001. Figure 2. Targeted deletion of Btg1 cooperates with ETV6-RUNX1 in regulating critical effector pathways implicated in leukemia. Relative expression levels of Bcl6, Tp53 and p19arf in empty control (Ctrl) and ETV6-RUNX1 transduced wild-type and Btg1-deficient fetal liver-derived hematopoietic progenitor cells by real-time PCR and normalized to the expression of the housekeeping gene TATA box binding protein (TBP). Data represent the mean and SEM of three independent experiments. *, P〈 0.05, **, P〈 0.01, ***, P〈 0.001. Disclosures No relevant conflicts of interest to declare.