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: 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.

Description: Single copy deletions of IKZF1, which occur in 10-15% of all B cell precursor acute lymphoblastic leukemia (BCP-ALL) cases, are associated with a poor outcome. We previously showed that loss of IKZF1 dictates resistance to glucocorticoids (GC) in BCP-ALL cell lines, a knockout mouse model and ex-vivo analysis of primary leukemic cells. When we analyzed the initial response to prednisolone therapy, we found that pediatric patients who suffer from an IKZF1 deleted leukemia are strongly enriched in the poor responder group (14 vs 7%, p

Description: Deletions and mutations affecting transcription factor IKZF1 are associated with increased relapse risk and poor outcome in B cell precursor acute lymphoblastic leukemia (BCP-ALL). However, additional genetic events may either enhance or negate the effects of IKZF1 on prognosis. We observed that deletions of the gene encoding the transcriptional coregulator BTG1 frequently co-occured with loss of IKZF1 function, suggesting a synergistic role for these events during leukemia development or progression. Targeted deletion of Btg1 predisposed both Btg1+/- and Btg1-/- mice to T cell malignancies, similar to what has been observed in Ikzf1 heterozygous knockout animals. Hence, while somatic single single-copy losses of either BTG1 and IKZF1 in the patient are predominantly found in BCP-ALL, targeted deletion of these genes in the mouse gives rise to T cell malignancies. To establish whether loss of BTG1 function affected the tumor suppressive role of IKZF1, the Btg1 knockout allele was crossed onto mice heterozygous for a loss-of-function Ikzf1 allele. Leukemia penetrance in these compound mice increased in a Btg1 dose-dependent manner. These leukemias were characterized by clonal TCRb rearrangement and aggressive infiltration into secondary organs, indicating synergistic roles for these tumor suppressors during mouse leukemia development. To investigate the effects of combined IKZF1/BTG1 loss in human BCP-ALL, we examined a large pediatric cohort of BCP-ALL cases, and found that the combined presence of BTG1 and IKZF1 deletions was associated with a markedly higher incidence of relapse, relative to IKZF1-deleted cases without BTG1 aberrations. Similar to BTG1 copy number losses, deletions in EBF1, PAX5, RB1 and CDKN2A/B appeared to be selectively enriched in IKZF1 deleted ALL. However, in contrast to BTG1, none of these other copy number alterations affected relapse incidence or outcome in this patient group. In conclusion, our data demonstrate synergy between the tumor suppressors BTG1 and IKZF1 during mouse leukemia development while the combined (single copy) loss of these two tumor suppressors identifies a patient group with an extremely poor outcome. Event free survival in a cohort of 514 children newly diagnosed with BCP-ALL, divided into four categories based on IKZF1 and BTG1 deletion status. Figure 1. Combined loss of IKZF1 and BTG1 predicts poor outcome in BCP-ALL Figure 1. Combined loss of IKZF1 and BTG1 predicts poor outcome in BCP-ALL Disclosures Pieters: Eusa Pharma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.