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: Abstract 1104 Poster Board I-126 Relapse is the most common cause of treatment failure in pediatric acute lymphoblastic leukemia (ALL), and is difficult to predict from information at diagnosis in the majority of cases. To explore the prognostic impact of recurrent copy number abnormalities on relapse in children diagnosed with precursor-B cell ALL, we performed genome-wide copy number profiling of 34 paired diagnosis-relapse samples. Lesions detected at diagnosis were often absent at relapse, including recurrent targets in precursor-B ALL like PAX5 (not preserved in 2 out of 7 cases with deletions at diagnosis), CDKN2A (not preserved in 1 out of 15 cases), and EBF (not preserved in 2 out of 5 cases), which illustrates that these lesions are often secondary events that are not present in the therapy-resistant progenitor that causes relapse. In contrast, deletions and nonsense mutations in IKZF1, which encodes the lymphoid differentiation factor IKAROS, were highly frequent (38%) and always preserved at time of relapse. Locus-specific copy number screening of IKZF1 in an additional cohort of diagnosis samples from children enrolled in the Dutch treatment protocol DCOG-ALL9 with (n=40) or without (n=51) relapse revealed that IKZF1 deletions were significantly enriched in relapse-prone cases (22.5% vs 3.9%; P=0.007). An independent and unbiased validation cohort of 150 DCOG-ALL9 cases was used to confirm these findings, which established that 28.6% of the cases with IKZF1 deletion at diagnosis developed a relapse. Together, we conclude that deletions of IKZF1 in DCOG-ALL9 treated pediatric precursor-B ALL patients provide a strong prognostic marker for relapse. Disclosures No relevant conflicts of interest to declare.

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.