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Chromosomal translocations in cancer genomes, key players in many types of cancers, generate chimeric proteins that drive oncogenesis. Genomes with chromosomal rearrangements can also produce fusion circular RNAs (f-circRNAs) by backsplicing of chimeric transcripts, as first shown in leukemias with PML::RARα and KMT2A::MLLT3 translocations and later in solid cancers. F-circRNAs contribute to the oncogenic processes and reinforce the oncogenic activity of chimeric proteins. In leukemia with KMT2A::AFF1 (MLL::AF4) fusions, we previously reported specific alterations of circRNA expression, but nothing was known about f-circRNAs. Due to the presence of two chimeric sequences, fusion and backsplice junctions, the identification of f-circRNAs with available tools is challenging, possibly resulting in the underestimation of this RNA species, especially when the breakpoint is not known. We developed CircFusion, a new software tool to detect linear fusion transcripts and f-circRNAs from RNA-seq data, both in samples for which the breakpoints are known and when the information about the joined exons is missing. CircFusion can detect linear and circular chimeric transcripts deriving from the main and reciprocal translocations also in the presence of multiple breakpoints, which are common in malignant cells. Benchmarking tests on simulated and real datasets of cancer samples with previously experimentally determined f-circRNAs showed that CircFusion provides reliable predictions and outperforms available methods for f-circRNA detection. We discovered and validated novel f-circRNAs in acute leukemia harboring KMT2A::AFF1 rearrangements, leading the way to future functional studies aimed to unveil their role in this malignancy.

Covalent Bruton tyrosine kinase (BTK) inhibitors, such as ibrutinib, have proven to be highly beneficial in the treatment of chronic lymphocytic leukemia (CLL). Interestingly, the off-target inhibition of IL-2-inducible T-cell kinase (ITK) by ibrutinib may also play a role in modulating the tumor microenvironment, potentially enhancing the treatment benefit. However, resistance to covalently binding BTK inhibitors can develop as the result of a mutation in cysteine 481 of BTK (C481S), which prevents irreversible binding of the drugs. In the present study we performed preclinical characterization of vecabrutinib, a next-generation noncovalent BTK inhibitor that has ITK-inhibitory properties similar to those of ibrutinib. Unlike ibrutinib and other covalent BTK inhibitors, vecabrutinib showed retention of the inhibitory effect on C481S BTK mutants in vitro, similar to that of wild-type BTK. In the murine Eμ-TCL1 adoptive transfer model, vecabrutinib reduced tumor burden and significantly improved survival. Vecabrutinib treatment led to a decrease in CD8+ effector and memory T-cell populations, whereas the naive populations were increased. Of importance, vecabrutinib treatment significantly reduced the frequency of regulatory CD4+ T cells in vivo. Unlike ibrutinib, vecabrutinib treatment showed minimal adverse impact on the activation and proliferation of isolated T cells. Lastly, combination treatment with vecabrutinib and venetoclax augmented treatment efficacy, significantly improved survival, and led to favorable reprogramming of the microenvironment in the murine Eμ-TCL1 model. Thus, noncovalent BTK/ITK inhibitors, such as vecabrutinib, may be efficacious in C481S BTK mutant CLL while preserving the T-cell immunomodulatory function of ibrutinib.

We previously identified an association of rapid engraftment of patient-derived leukemia cells transplanted into NOD/SCID mice with early relapse in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). In a search for the cellular and molecular profiles associated with this phenotype, we investigated the expression of microRNAs (miRNAs) in different engraftment phenotypes and patient outcomes. We found high expression of miR-497 and miR-195 (hereafter miR-497/195) in patient-derived xenograft samples with slow engraftment derived from patients with favorable outcome. In contrast, epigenetic repression and low expression of these miRNAs was observed in rapidly engrafting samples associated with early relapse. Overexpression of miR-497/195 in patient-derived leukemia cells suppressed in vivo growth of leukemia and prolonged recipient survival. Conversely, inhibition of miR-497/195 led to increased leukemia cell growth. Key cell cycle regulators were downregulated upon miR-497/195 overexpression, and we identified cyclin-dependent kinase 4 (CDK4)– and cyclin-D3 (CCND3)–mediated control of G1/S transition as a principal mechanism for the suppression of BCP-ALL progression by miR-497/195. The critical role for miR-497/195–mediated cell cycle regulation was underscored by finding (in an additional independent series of patient samples) that high expression of miR-497/195 together with a full sequence for CDKN2A and CDKN2B (CDKN2A/B) was associated with excellent outcome, whereas deletion of CDKN2A/B together with low expression of miR-497/195 was associated with clearly inferior relapse-free survival. These findings point to the cooperative loss of cell cycle regulators as a new prognostic factor indicating possible therapeutic targets for pediatric BCP-ALL.

Circular RNAs (circRNAs) are abundantly expressed in the haematopoietic compartment, but knowledge on their diversity among blood cell types is still limited. Nevertheless, emerging data indicate an array of circRNA functions exerted through interactions with other RNAs and proteins, by translation into peptides, and circRNA involvement as regulatory molecules in many biological processes and cancer mechanisms. Interestingly, the role of specific circRNAs in leukemogenesis has been disclosed by a few studies, mostly in acute myeloid leukemia. In this study, circRNA expression in B-cells, T-cells and monocytes of healthy subjects is described, including putative new circRNA genes. Expression comparison considered 6,228 circRNAs and highlighted cell population-specific expression and exon usage patterns. Differential expression has been confirmed by qRT-PCR for circRNAs specific of B-cells (circPAX5, circAFF3, circIL4R, and circSETBP1) or T-cells (circIKZF1, circTNIK, circTXK, and circFBXW7), and for circRNAs from intronic (circBCL2) and intergenic regions that were overexpressed in lymphocytes. Starting from this resource of circRNA expression in mature blood cell populations, targeted examination identified striking and generalized upregulated expression of circPAX5, circPVT1 and circHIPK3 in pediatric B-precursor acute lymphoblastic leukemia, and disclosed circRNAs with variable expression across cytogenetic subtypes.

Chromosomal translocations harbored by cancer genomes are important oncogenic drivers. In MLL rearranged acute leukemia (MLLre) MLL/KMT2A fuses with over 90 partner genes. Mechanistic studies provided clues of MLL fusion protein leukemogenic potential, but models failed to fully recapitulate the disease. Recently, expression of oncogenic fusion circular RNAs (f-circ) by MLL-AF9 fusion was proven. This discovery, together with emerging data on the importance and diversity of circRNAs formed the incentive to study the circRNAs of the MLL recombinome. Through interactions with other RNAs, such as microRNAs, and with proteins, circRNAs regulate cellular processes also related to cancer development. CircRNAs can translate into functional peptides too. MLL and most of the 90 MLL translocation partners do express circRNAs and exploration of our RNA-seq dataset of sorted blood cell populations provided new data on alternative circular isoform generation and expression variability of circRNAs of the MLL recombinome. Further, we provided evidence that rearrangements of MLL and three of the main translocation partner genes can impact circRNA expression, supported also by preliminary observations in leukemic cells. The emerging picture underpins the view that circRNAs are worthwhile to be considered when studying MLLre leukemias and provides a new perspective on the impact of chromosomal translocations in cancer cells at large.

Chromosomal translocations harbored by cancer genomes are important oncogenic drivers. In MLL rearranged acute leukemia (MLLre) MLL/KMT2A fuses with over 90 partner genes. Mechanistic studies provided clues of MLL fusion protein leukemogenic potential, but models failed to fully recapitulate the disease. Recently, expression of oncogenic fusion circular RNAs (f-circ) by MLL-AF9 fusion was proven. This discovery, together with emerging data on the importance and diversity of circRNAs formed the incentive to study the circRNAs of the MLL recombinome. Through interactions with other RNAs, such as microRNAs, and with proteins, circRNAs regulate cellular processes also related to cancer development. CircRNAs can translate into functional peptides too. MLL and most of the 90 MLL translocation partners do express circRNAs and exploration of our RNA-seq dataset of sorted blood cell populations provided new data on alternative circular isoform generation and expression variability of circRNAs of the MLL recombinome. Further, we provided evidence that rearrangements of MLL and three of the main translocation partner genes can impact circRNA expression, supported also by preliminary observations in leukemic cells. The emerging picture underpins the view that circRNAs are worthwhile to be considered when studying MLLre leukemias and provides a new perspective on the impact of chromosomal translocations in cancer cells at large.

Central nervous system acute lymphoblastic leukemia (CNS-ALL) is a major clinical problem. Prophylactic therapy is neurotoxic, and a third of the relapses involve the CNS. Increased expression of interleukin 15 (IL-15) in leukemic blasts is associated with increased risk for CNS-ALL. Using in vivo models for CNS leukemia caused by mouse T-ALL and human xenografts of ALL cells, we demonstrate that expression of IL-15 in leukemic cells is associated with the activation of natural killer (NK) cells. This activation limits the outgrowth of leukemic cells in the periphery, but less in the CNS because NK cells are excluded from the CNS. Depletion of NK cells in NOD/SCID mice enabled combined systemic and CNS leukemia of human pre-B-ALL. The killing of human leukemia lymphoblasts by NK cells depended on the expression of the NKG2D receptor. Analysis of bone marrow (BM) diagnostic samples derived from children with subsequent CNS-ALL revealed a significantly high expression of the NKG2D and NKp44 receptors. We suggest that the CNS may be an immunologic sanctuary protected from NK-cell activity. CNS prophylactic therapy may thus be needed with emerging NK cell-based therapies against hematopoietic malignancies.

Juvenile myelomonocytic leukemia (JMML) is a rare aggressive disease of early childhood. Driver mutations in the Ras signaling pathways are a key feature of JMML patients. Mutations in SETBP1 and JAK3 were recently identified in a subset of JMML patients characterized by poor prognosis and progression of disease. In this study, we report the results of a screening for mutations in SETBP1 and JAK3 of a cohort of seventy Italian patients with JMML, identifying 11.4% of them harboring secondary mutations in these two genes and discovering two new mutations in the SKI domain of SETBP1. JMML xenotransplantation and colony assay provide an initial understanding of the secondary nature of these events occurring in early precursor cells and suggest a different propagating capacity of clones harboring particular mutations.

// Md. Nabiul Hasan 1 , Manon Queudeville 1 , Luca Trentin 1 , Sarah Mirjam Eckhoff 1 , Ilaria Bronzini 2 , Chiara Palmi 3 , Thomas Barth 4 , Giovanni Cazzaniga 3 , Geertruy te Kronnie 2 , Klaus-Michael Debatin 1 and Luder Hinrich Meyer 1 1 Department of Pediatrics and Adolescent Medicine, Ulm University Medical Center, Ulm, Germany 2 Department of Women’s and Children’s Health, University of Padova, Padova, Italy 3 Centro Ricerca Tettamanti, Clinica Pediatrica, University of Milano-Bicocca, Monza, Italy 4 Institute for Pathology, Ulm University Medical Center, Ulm, Germany Correspondence: Luder Hinrich Meyer, email: // Keywords : Acute lymphoblastic leukemia, pediatric, xenograft model, mTOR hyperactivation, preclinical targeting Received : September 03, 2014 Accepted : December 01, 2014 Published : December 02, 2014 Abstract Despite increasingly successful treatment of pediatric ALL, up to 20% of patients encounter relapse. By current biomarkers, the majority of relapse patients is initially not identified indicating the need for prognostic and therapeutic targets reflecting leukemia biology. We previously described that rapid engraftment of patient ALL cells transplanted onto NOD/SCID mice (short time to leukemia, TTL short ) is indicative of early patient relapse. Gene expression profiling identified genes coding for molecules involved in mTOR signaling to be associated with TTL short /early relapse leukemia. Here, we now functionally address mTOR signaling activity in primograft ALL samples and evaluate mTOR pathway inhibition as novel treatment strategy for high-risk ALL ex vivo and in vivo . By analysis of S6-phosphorylation downstream of mTOR, increased mTOR activation was found in TTL short /high-risk ALL, which was effectively abrogated by mTOR inhibitors resulting in decreased leukemia proliferation and growth. In a preclinical setting treating individual patient-derived ALL in vivo , mTOR inhibition alone, and even more pronounced together with conventional remission induction therapy, significantly delayed post-treatment leukemia reoccurrence in TTL short /high-risk ALL. Thus, the TTL short phenotype is functionally characterized by hyperactivated mTOR signaling and can effectively be targeted ex vivo and in vivo providing a novel therapeutic strategy for high-risk ALL.

In acute lymphoblastic leukemia (ALL) leukemia initiating cells (LICs) have been considered to be organized in an hierarchical fashion, however recent findings demonstrated LIC-activity also in more committed cells supporting a stochastic stem cell concept. Thus, the nature of leukemia initiating cells in ALL still remains elusive. As an alternative approach to define LICs by expression of cellular markers as commonly employed, we addressed LIC activities in ALL by functional investigation of cellular subfractions of distinct cell cycle phases. Patient-derived xenograft BCP-ALL cells were sorted according to cell cycle stages (i.e. G0/G1 and G2/M) and subsequently transplanted onto NOD/SCID mice. All cell fractions led to engraftment indicating LIC activity of all leukemia cells. However, cells isolated from G0/G1 cell cycle phases led to early leukemia onset in contrast to cells from late cell cycle (G2/M) constantly showing lowest LIC activity. Strikingly, this difference in LIC activity was maintained upon secondary transplantation. In an alternative approach, we investigated metabolic activities in cellular leukemia subfractions and identified low metabolic activity in cells of early G0/G1 cell cycle phases compared to increased cellular metabolism in cells of late G2/M. To address LIC-capacities of ALL cells with distinct metabolic activities on a functional level, cellular fractions were sorted according to low or high ROS levels and subsequently transplanted onto NOD/SCID mice. Interestingly, a prolonged engraftment was observed upon transplantation of ROShigh cells in contrast to faster leukemia repopulation in recipients transplanted with ROSlow cells, showing that the metabolic activity is indicative for its leukemia initiating activity. In summary, we identified LIC-activity in all leukemia subpopulations. Importantly, our findings indicate that leukemia initiating cells in ALL are enriched in early cell cycle and characterized by low metabolic activity.

Over the past decade, xenografting human leukemia cells into mice with different levels of immunodeficiency, with or without preconditioning, has provided an important tool to study various aspects of leukemia biology and to identify distinct clinical risk groups for evaluation of novel therapeutic strategies, as well as the possibility of amplifying human leukemia cells in vivo. Interestingly, these models using human acute lymphoblastic leukemia and acute myeloid leukemia cells as xenografts recapitulate many clinical features of the disease. Similar to the human environment (for example, in the bone marrow), transplanted leukemia cells in the murine setting are exposed to both favorable and unfavorable conditions for engraftment that may exert a distinct pressure for selection of subclones. Thus, results obtained in these models may vary depending on the experimental setup. The impact of in vivo growth of human leukemia cells on the background of a more or less hostile murine environment for leukemia biology and the course of the disease in patients are discussed in the context of the diversity of xenograft models. Cancer Res; 71(23); 7141–4. ©2011 AACR.

Abstract 1629 Poster Board I-655 Primary childhood acute lymphoblastic leukemia (ALL) samples are very difficult to culture in vitro and the currently available cell lines only poorly reflect the heterogeneous nature of the primary disease. Many groups therefore use mouse xenotransplantation models not only for in vivo testing but also as a means to amplify the number of leukemia cells to be used for various analysis. It remains unclear as to what extent the xenografted samples recapitulate their respective primary leukemia. It has been suggested for example that transplantation may result in the selection of a specific clone present only to a small amount in the primary diagnostic sample. We used a NOD/SCID xenotransplantation model and injected leukemia cells isolated from fresh primary diagnostic material of 4 pediatric ALL patients [2 pre-B-ALL, 1 pro-B-ALL (MLL/AF4}, 1 cortical T-ALL] intravenously into the lateral tail vein of unconditioned mice. As soon as the mice presented clinical signs of leukemia, leukemia cells were isolated from bone marrow and spleen. Isolated leukemia cells were retransplanted into secondary and tertiary recipients. RNA was isolated from diagnostic material and serial xenograft passages and gene expression profiles were obtained using a human whole genome array (Affymetrix U133 2.0). Simultaneously, immunophenotypic analysis via multicolor surface and cytoplasmatic staining by flow cytometry was performed for the diagnostic samples and respective serial xenograft passages. In an unsupervised clustering analysis the diagnostic sample of each patient clustered together with the 3 derived xenograft samples, although the 3 xenograft samples clustered stronger to each other than to their respective diagnostic sample. Comparison of the 4 diagnostic samples vs. all xenograft samples resulted in a gene list of 270 genes upregulated at diagnosis and 8 genes upregulated in the xenograft passages (Wilcoxon, p< .05). The high number of genes upregulated at diagnosis is most likely due to contamination of primary patient samples with normal peripheral blood and/or bone marrow cells as 15% of genes are attributed to myeloid cells, 7% to erythroid cells, 7% to lymphoid cells, 32% to bone marrow in general as well as to innate immunity, chemokines, immunoglobulins. The remaining genes can not be attributed to a specific hematopoetic cell lineage and are not known to be related to leukemia or cancer in general. Accordingly, there are no statistically significant differences between the primary, secondary and tertiary xenograft passages. The immunophenotype analysis are also in accordance with these findings, as the diagnostic blast population retains its immunophenotypic appearance during serial transplantation, whereas the contaminating CD45-positive non- leukemic cells disappear after the first xenograft passage. The few genes upregulated in xenograft samples compared to diagnosis are mainly involved in cell cycle regulation, protein translation and apoptosis resistance. Some of the identified genes have already been described in connection with cancer subtypes, their upregulation therefore being indicative of a high proliferative state in general and could argue towards a more aggressive potential of the engrafted leukemia cells but alternatively could also simply be due to the fact that the xenograft samples are pure leukemic blasts and are not contaminated with up to 15% of non-cycling healthy bone marrow cells as in the diagnostic samples. We conclude that the gene expression profiles generated from xenografted leukemias are very similar to those of their respective primary leukemia and moreover remain stable over serial retransplantation passages as we observed no statistically significant differences between the primary, secondary and tertiary xenografts. The differentially expressed genes between diagnosis and primary xenotransplant are most likely to be due to contaminating healthy cells in the diagnostic samples. Hence, the NOD/SCID-xenotransplantation model recapitulates the primary human leukemia in the mouse and is therefore an appropriate tool for in vivo and ex vivo studies of pediatric acute leukemia. Disclosures No relevant conflicts of interest to declare.

Over the past decades, the use of optimized multi-agent chemotherapy protocols and improved risk stratification have led to superior patient outcome. However, avoiding therapy-related toxicity, decreasing the relapse rate of currently almost 20 % and improving the outcome of relapsed patients remain challenging problems. Aberrant activity of pathways involved in the regulation of survival and cell death contribute to development of leukemia and therapy failure. Anti-apoptotic BCL-2 family proteins are key regulators of apoptosis providing a promising target for novel directed therapies. The BH3 mimetic ABT-199 binds to BCL-2, counteracts its anti-death function and leads to apoptosis induction by direct release of pro-death BCL-2 family proteins such as BAX. BH3 mimetics are currently used pre-clinically and in first clinical trials. However the intrinsic or acquired resistance indicates the need for predictive markers and for effective combination treatment strategies. In this study, we addressed the activity of ABT-199 in B cell precursor (BCP) ALL. The effects of ABT-199 as single agent and in combination with chemotherapeutic drugs used in remission-induction therapy of pediatric ALL were analyzed using BCP-ALL cell lines (n=5) and a set of patient-derived primograft samples (n=9) established by transplantation of primary leukemia cells obtained from pediatric BCP-ALL patients at diagnosis onto immunodeficient NOD/SCID mice. Since ABT-199 does not bind to all antiapoptotic BCL-2 family members, sparing MCL-1, we included the cyclin-dependent kinase (CDK) inhibitor dinaciclib, which also exerts MCL-1 inhibitory effects. Cell viability was determined by flowcytometry according to fw/sc criteria and half maximal inhibitory concentrations (IC50) were estimated upon exposure to single agents and drug combinations. Combination indices (CI) indicating synergistic or additive effects were estimated according to Chou-Talalay. BCL-2 and MCL-1 protein levels were investigated by western blot analysis. Despite sensitivity in most of the BCP-ALL cell lines with IC50 values in the nanomolar range (mean IC50, 212 nM), one cell line, Nalm-6, showed ABT-199 resistance with an IC50 >10 µM. Interestingly, low protein levels of BCL-2 and increased MCL-1 expression were identified in this insensitive line, in contrast to high level BCL-2 and low MCL-1 expression in ABT-199 sensitive leukemias, indicating a potential marker to identify sensitive and resistant ALL. A pronounced synergism of ABT-199 in combination with chemotherapeutic agents was found with vincristine (CI 0.31 and 0.40, cell lines RS4;11 and Nalm-6) and even strong synergism with asparaginase and dexamethasone in ABT-199 monotherapy resistant Nalm-6 cells (CI 0.002, 0.14). Strikingly, the combination of the MCL-1 inhibitor dinaciclib with ABT-199 was highly effective and revealed a strong synergism for both compounds (CI 0.03). When we addressed the effects of ABT-199 on primograft ALL samples isolated from mice with full-blown leukemia, a pattern of high sensitivity (IC50 from 16 to 156 nM) similar to the cell lines was observed. In contrast, peripheral blood (PB) samples from healthy control donors were resistant to ex vivo exposure with ABT-199 (n=3, IC50 values > 1 µM). However, one primograft sample derived from a high risk patient showed ABT-199 insensitivity with an IC50 value of > 1 µM similar to healthy PBMCs. This primograft sample was also characterized by low BCL-2/high MCL-1 protein expression similar to Nalm-6. Most importantly, this resistant leukemia was also rendered ABT-199 sensitive by co-treatment with dinaciclib (CI 0.04). Taken together, we found efficacy of ABT-199 in the majority BCP-ALL cell lines and patient-derived primograft ALL samples and ABT-199 synergized with conventional chemotherapeutic agents. ABT-199 resistant leukemias characterized by low BCL-2/high MCL-1 expression were resensitized by addition of dinaciclib leading to strong synergism. These data indicate effective cell death sensitization by ABT-199 and potential strategies to overcome ABT-199 resistance in BCP-ALL. Disclosures No relevant conflicts of interest to declare.

B Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL) represents 35% of all cancers in pediatric age group. The cure rate for this disease approaches 90% with current treatment regimens, however only a third of patients with relapse are cured. Therefore, there is an urgent need to focus on subgroups of patients with hallmarks of bad prognosis that could benefit from novel therapeutic approaches. Alterations of Cytokine Receptor-like Factor 2 (CRLF2), a negative prognostic factor in pediatric BCP-ALL, have been identified in up to 10% of patients. However these patients represent half of the high risk Ph-like ALL and of Down Syndrome-associated BCP-ALL. Rearrangements of CRLF2 result in the overexpression of this component of the heterodimeric cytokine receptor for thymic stromal lymphopoietin (TSLP) and is associated with activating mutations of the JAK-STAT pathway. Together these cause hyperactivation of JAK/STAT and PI3K/mTOR signaling. Inhibition of CRLF2/JAK2 signaling has the potential to become a therapeutic targeted intervention for this subgroup of poor prognostic patients. Previous studies have shown that the HDAC inhibitor Givinostat/ITF2357 has potent anti-tumor activity against hematological malignancies, particularly JAK2V617F mutated myeloproliferative neoplasms (MPN) such as polycythemia vera, for which it has already a clinic application and established safety profile. We therefore studied the in vitro and in vivo efficacy of Givinostat in cases with CRLF2 rearrangements. Here we demonstrated that Givinostat inhibited proliferation and induced apoptosis of BCP-ALL CRLF2-rearranged MHH-CALL4 and MUTZ5 cell lines positive for exon 16 JAK2 mutations. Of note, the observed IC50 values for MHH-CALL4 were lower than those for the SET2 cell line positive control bearing JAK2V617F mutation, both for proliferation (IC50: 0.08±0.05µM vs. 0.14±0.03µM) and apoptosis (IC50: 0.17±0.03µM vs. 0.22±0.04µM). We next investigated the effect of Givinostat on blasts from CRLF2 rearranged BCP-ALL patient samples. For this purpose we developed xenograft models of human CRLF2 rearranged ALL to expand cells from patients and to recapitulate human leukemia in recipient mice. ALL blasts isolated from xenografts were co-cultured on OP9 stroma to perform ex vivo assays. Consistent with our findings in cell lines, Givinostat (0.2µM) reduced the % of live cells (Annexin V/Sytox negative) in all xenografts treated with the drug. In particular, after 72 hours, Givinostat was able to kill up to >90% of blast cells in all xenografts in contrast with the vehicle-treated samples which showed 25-60% of blasts still alive after treatment. The induction of cell death in Givinostat treated primografts was confirmed on primary samples from diagnosis using CyTOF which allowed us to observe that CD10+/CRLF2+ blasts were preferentially killed by the drug whereas CD45 high expressing cells (normal residue) remained unaffected by the treatment. Moreover, at low doses (0.2 µM), Givinostat downregulated genes of the JAK/STAT pathway (STAT5A, JAK2, IL7Rα, CRLF2, BCL2L1 and cMYC) and inhibited the basal and ligand induced signaling, reducing the phoshporylation of STAT5 in all tested primografts (mean fold decrease of pSTAT5: 2.4+0.6). Most importantly, to understand if the transcriptional downregulation of CRLF2 resulted in a functional effect, the downmodulation of CRLF2 protein was observed by flow cytometry (mean fold decrease 3.55+1.38). In vivo, Givinostat significantly reduced engraftment of human blasts in xenograft models of CRLF2 positive BCP-ALL (ranging from 1.9 to 34 fold decrease in bone marrow). Furthermore, Givinostat augmented the effect of chemotherapy in inhibiting proliferation and inducing apoptosis in CRLF2 rearranged cell lines and in primografts, in vitro. After 72 hours, the combined treatment reached 4.6-8.8 fold lower % of remaining viable blasts than chemotherapy alone (6.3-35.3% viable cells in chemotherapy-treated samples vs 1.4-4.3% of combination), 2.5-8.5 fold lower than Givinostat alone (4.3-36.4% vs 1.4-4.3%) and 2.4-13 fold lower than Methyl-prednisolone (5.2-39.1 vs 1-16.3%). In conclusion, Givinostat may represent a novel and effective tool, in combination with current chemotherapy, to treat this difficult to handle subset of ALL and these data strongly argue for the translation of Givinostat in combination with conventional therapy into human trials. Disclosures Davis: Fluidigm, Inc: Honoraria. Nolan:Fluidigm, Inc: Equity Ownership.

Acute lymphoblastic leukemia (ALL) is the most common childhood malignancy accounting for 80% of leukemias. The involvement of the central nervous system (CNS) by ALL is a major clinical problem and occurs in about 50% of the children without adequate treatment. The introduction of CNS-directed therapy consisting of intrathecal and high dose systemic chemotherapy and, occasionally, cranial irradiation, reduced relapse rate to less than 5% and has become a prerequisite for treating children with ALL. However, substantial neurotoxicity associated with this therapy is a major concern. Moreover the CNS is involved in up to a third from all relapses. To date very little is known about the pathogenesis of CNS leukemia. Our research was promoted by the previous observation that high mRNA expression of interleukin 15 (IL15) in leukemic blasts is associated with increased risk for CNS involvement (Cario et al JCO 2007;25:4813-20). As IL15 is a strong stimulant of Natural Killer (NK) cells, we hypothesized that the increased expression of IL15 may activate NK cells which, in turn, will control residual ALL cells in the peripheral blood but not in the relatively protected central nervous system. To investigate this hypothesis, we utilized two mouse models, a S49-derived T lymphoblastic leukemia syngeneic model and a novel human xenograft ALL model in immune-deficient mice. We found that constitutive expression of IL15 in mouse T lymphoblastic leukemia cells transplanted in neonatal Balb/c mice markedly slowed the development of systemic disease and caused CNS leukemia characterized by pronounced clinical CNS symptoms and subarachnoid infiltration of leukemia cells. This phenotype was accompanied by increase in activated natural killer (NK) cells (from 0.16% to 18.6% P Taken together we show here, for the first time, a crucial role for NK cells in the control of CNS leukemia. We suggest that the association between IL15 expression in ALL blasts and isolated CNS relapse might be explained by activation of NK cells leading to increased surveillance of residual leukemia in the bone marrow but not in the CNS which serve as a sanctuary site for tumor growth. Disclosures: No relevant conflicts of interest to declare.

Abstract 2985 Recently, we identified in a NOD/SCID human (hu) B-cell precursor (BCP) acute lymphoblastic leukemia (ALL) mouse model two different engraftment phenotypes that we named time to leukemia (TTL) short (TTLshort) and TTL long (TTLlong) and that reflected the rapid or late onset of leukemia in the mice. We showed that the rapid and late leukemia engraftments in the mice highly correlated with the relapse free survival of patients initially stratified as standard risk at diagnosis. In order to analyze if the two distinct phenotypes are characterized by different frequency of leukemia initiating cells (LICs), we investigated the NOD/SCID repopulating activity of 4 individual BCP ALL samples. We performed a limiting dilution transplantation assay using freshly isolated spleen cells form 2 TTLshort and 2 TTLlong xenografted mice with full-blown leukemia. We created 5 groups of 8 mice each and transplanted intravenously 105, 104, 103, 102, 101 cells per mouse/group. Leukemia engraftment in the peripheral blood (PB) was routinely evaluated by flowcytometry and the mice were sacrificed at the onset of disease manifestation. We defined a mouse as engrafted when we could detect ≥ 1% CD19 positive cells in the PB. In 3 out of 4 samples, 100% of the mice transplanted with 105 and 104 cells engrafted; only in one sample (TTLlong) we had a 75% and 12,5% successful engraftment using 105 and 104 cells respectively. The percentage of engrafted mice using 103 cells was 87,5% and 75% for the 2 TTLshort samples and 37,5% and 0% for the 2 TTLlong specimens. No mice transplanted with the 102 cells isolated from the 2 TTLlong samples engrafted whereas 50% and 12,5% of the mice transplanted with the 102 cells isolated from the 2 TTLshort engrafted successfully. No one of the mice transplanted with 101 cells engrafted. The LIC frequency was calculated using the Poisson single-hit model; a higher LIC frequency was calculated in the 2 TTLshort (1/329 and 1/739) compared to the 2 TTLlong (1/2159 and 1/74028). As cells in the G0 phase of the cell cycle are considered as the reservoir for the new cycling cells and putative LICs, we analyzed by flowcytometry the percentage of G0 cells, defined as cells staining negative for the Ki-67 antigen, within TTLshort (N=8) and TTLlong (N=7). No statistical difference was found between the analyzed samples. Nevertheless, we observed that in both TTLshort and TTLlong almost all cells were actively progressing through the cell cycle. Aiming to further characterize distinctive biological features related to the different LIC frequency, we analyzed by flowcytometry the cell cycle profiles of 9 TTLshort and 8 TTLlong mice. We used a cell cycle analysis based on the simultaneous labeling of DNA (7-AAD) and RNA (Pyronin Y) that allows to distinguish between cells that are in G1 but have not yet actively entered the S phase of the cell cycle (named G1a cells) from cells that are actively progressing form G1 to S (named G1b cells). A higher percentage of G1a cells was detected in the TTLshort compared to TTLlong (p = 0.004 Mann-Whitney Test) suggesting the presence of a stand-by-cell fraction in the TTLshort almost ready to proceed through the cell cycle. On the other hand, a higher percentage of G1b cells was found in the TTLlong compared to TTLshort (p = 0.001, Mann-Whitney Test). No significant difference was found in the proportion of cells in the “S/G2/M subgroup”. We also analyzed by Western Blot CYCLIN B1 expression in TTLshort (N=5) and TTLlong (N=5): a statistically different CYCLIN B1 expression was found between the two groups (p = 0.009 Mann-Whitney Test). As this molecule is involved in the progression of cells from the G2 to M phase of the cell cycle, we analyzed by flowcytometry the mitotic cells fraction defined as the cells positive for the phosphorilation of the Ser10 on the histone H3 in 6 TTLshort and 5 TTLlong specimens. A higher proportion of P-H3(Ser10) positive cells was detected in the TTLshort compared to the TTLlong (p = 0.045, Mann-Whitney Test). All together these data indicate that the two NOD/SCID engraftment phenotypes are characterized by different frequency of LICs. Furthermore, our functional analyses reveal a distinctive progression of the leukemic cells through the cell cycle showing a higher number of cells (G1a cells) ready to progress through the G1-S phases in the TTLshort/poor prognosis leukemia. Disclosures: No relevant conflicts of interest to declare.

Abstract 1457 Acute lymphoblastic leukemia (ALL) is the most frequent form of childhood cancer. Although therapy efforts have achieved cure rates of above 80%, about every fifth patient encounters relapse, which is associated with poor outcome. Risk stratification based on prognostic factors is critical for selecting appropriate treatment intensity to improve therapy efficacy and reduce toxicity. However, in spite of new strategies for risk stratification, the majority of relapsed patients are initially stratified into non-high risk groups pointing out the need to identify new prognostic markers that might refer to novel therapeutic targets. We have recently shown in a NOD/SCID/huALL mouse xenotranplant model that early relapse of pediatric B-cell precursor (BCP-) ALL patients is reflected by rapid engraftment (time to leukemia/ TTLshort) of primary ALL cells. In this study we aimed to identify differently expressed and activated proteins in xenograft ALL of distinct engraftment subgroups employing a Reverse Phase Protein Array (RPPA) strategy. Levels of total or activated proteins were analyzed in patient derived xenograft BCP-ALL samples characterized with respect to their NOD/SCID engraftment phenotype (TTLshort n= 7, TTLlong n= 9). The proteome of each sample was immobilized on nitrocellulose coated glass slides and overall protein expression or phosphorylation of 51 key signaling molecules was detected by incubation with the corresponding specific antibodies. Protein expression and/ or activation was quantified and compared between the different engraftment subgroups (TTLshort/ early relapse versus TTLlong/ good prognosis). RPPA results were validated by Western Blot analyses. The association of NOD/SCID/huALL engraftment and clinical patient outcome was analyzed and revealed a significantly inferior survival of patients with the TTLshort in contrast to the TTLlong phenotype also in this subgroup of patients (Kaplan Meier, log rank, P=.003). Upon comparison of the protein expression data (Shrinkage t-test, P CYCLIN B is a positive regulator of the cell cycle and highly expressed in the G2/M phase. Consistent with its pro-proliferative function, CYCLIN B was identified to be up-regulated in leukemia samples with rapid NOD/SCID/huALL growth (TTLshort/ early relapse phenotype) (P=.013). Interestingly, the level of CYCLIN B protein expression correlates negatively to TTL (Spearman, rs=-.516, P=.041), suggesting a direct association of leukemia cell cycle progression and leukemia engraftment in the NOD/SCID/huALL xenograft model. Beta-CATENIN, a molecule involved in WNT-signaling and cell adhesion but also associated with apoptosis inhibition and cell growth in other hematological malignancies, was found to be over-expressed in TTLshort (P=.006). Furthermore, beta-CATENIN protein levels were reversely correlated to TTL (Spearman, rs=-.557, P=.025). Additionally, ANNEXIN I, involved in the regulation of inflammation and apoptosis and reported to be up-regulated in hairy cell leukemia, showed high protein levels in TTLshort (P=.033). Protein-kinase C alpha was previously reported to induce apoptosis in an ALL- cell line and showed increased activation in non-relapsing pediatric T-ALL patient samples. In line with this, we found higher levels of PKC alpha activation in the TTLlong -group (P= 8.5e–9), indicating an association with good prognosis. Taken together, this study identified differentially expressed proteins in prognostic subgroups of BCP-ALL patients with distinct clinical outcomes, which can be further evaluated as new prognostic markers and therapeutic targets. Disclosures: Debatin: Apogenix GmbH: Patents & Royalties, Research Funding.

Abstract 2572 Treatment of acute lymphoblastic leukemia (ALL), the most frequent malignant disease in children and adolescents, is increasingly successful. Stratification based on prognostic factors assigning the patients to treatment protocols of different intensity and intensification of chemotherapy regimens have improved patient outcome with present cure rates above 80%. However, about 20% of the patients suffer from relapse which is associated with inferior outcome, especially if occurring early. Importantly, despite the efforts achieved by stratification strategies including detection of minimal residual disease, the majority of relapse patients are initially stratified into non-high risk groups and not identified upfront by current markers. This clearly emphasizes the need for additional prognostic markers which should ideally reflect features of leukemia biology pointing to novel therapeutic targets. In a recent study we described a strong association of the engraftment phenotype observed in a series of transplanted primary patient B cell precursor (BCP) ALL samples in a NOD/SCID/huALL mouse model and early patient relapse. This rapid engraftment phenotype (time to leukemia, TTLshort) is characterized by a gene expression profile pointing to pathways involved in cell growth and proliferation. Since mammalian target of rapamycin (mTOR) is a key survival pathway and was identified in the gene profile we now investigated this pathway on a functional level. For this purpose, ALL xenograft samples previously characterized with respect to their engraftment phenotype (TTLshort/early relapse, n=3 or TTLlong/no or late relapse, n=4) were re-transplanted onto NOD/SCID mice. At disease onset ALL cells were harvested from leukemia bearing recipients and mTOR pathway activation was analyzed by flow cytometry assessing phosphorylation of ribosomal protein S6 (P-S6), a molecule downstream of mTOR. Increased P-S6 levels were detected in TTLshort compared to TTLlong xenografts (T-test, P=.009) indicating constitutive mTOR hyperactivation in TTLshort/early relapse leukemia. Furthermore, the effects of the mTOR inhibitor rapamycine and the PI3K/mTOR dual inhibitor BEZ235 were investigated. Xenograft leukemia cells were incubated with rapamycine, BEZ235 or solvent and P-S6 was analyzed. Interestingly, both inhibitors led to a significant decrease of pathway activation in TTLshort but not in TTLlong xenografts indicating that the hyperactivated mTOR pathway of this high risk ALL subtype can be successfully targeted. Moreover, the effect of mTOR inhibition on individual patient derived ALL was also analyzed in vivo. Since in a clinical setting high risk disease is unlikely to be treated employing a novel strategy on its own, we investigated the effect of mTOR inhibition in addition to multiagent drug treatment resembling ALL induction therapy. NOD/SCID mice were transplanted with TTLshort (n=2) or TTLlong (n=1) leukemia. Upon appearance of leukemia in the peripheral blood treatment with either rapamycine, multiagent chemotherapy, combination of chemotherapy and rapamycin or solvent was initiated (8 mice per group) and the time until reoccurrence of leukemia defined by presence of 25% or more human ALL cells in the recipients blood was assessed. In both TTLshort leukemias a significant delay of ALL onset was observed upon combination treatment compared to chemotherapy alone, however the TTLlong leukemia showed onset at similar time points irrespective of treatment modalities. Importantly, also a significant reduction in leukemia load (as measured by decreased spleen weights despite similar high percentage leukemia infiltration) was observed in TTLshort but not TTLlong leukemias upon in vivo combination therapy. Taken together, we demonstrated that TTLshort/early relapse leukemia is associated with an hyperactivated mTOR pathway and can effectively be targeted by mTOR inhibitors ex vivo. Most importantly, a significant delay of leukemia onset upon in vivo mTOR inhibition combined with conventional chemotherapy was achieved in a preclinical NOD/SCID/huALL xenograft model. Thus, mTOR inhibition is a successful novel therapeutic strategy for the treatment of high risk ALL. Disclosures: No relevant conflicts of interest to declare.

Abstract 2454 Mixed Lineage Leukemia (MLL) gene rearrangements are common genetic translocations occurring in human leukemia. Numerous MLL-partner genes have been identified so far; of these MLL-AF4 was found to characterize the largest subgroup of fusion proteins in pediatric acute lymphoblastic leukemia (ALL). A recent study reported that mutations in kras accelerate leukemo-lymphogenesis in a MLL-AF4 transgenic mouse model. Activating mutations in RAS genes, frequently occuring in codons 12, 13 and 61, prevent the hydrolysis of RAS-GTP and result in the constitutive activation of the RAS proteins enhancing cell survival and proliferation. Mutations in RAS genes were found in several types of human cancers, including leukemia and ALL with MLL rearrangements. We analyzed the presence of KRAS mutations in pediatric MLL-AF4 rearranged ALL samples at diagnosis and relapse with the aim to establish the frequency of KRAS mutations in MLL-AF4 leukemia and to tentatively associate RAS mutations to the leukemogenic process. In this study 40 ALL patients were included (23 infant and 17 non infant); of these, 14 pairs at diagnosis and relapse, 22 at diagnosis only and 4 samples at relapse only. The study was approved by the institutional ethical committee and informed consent was obtained in accordance with the Declaration of Helsinki. Mutation screening was performed for exon 2 and exon 3 of KRAS by 454 technology (Roche Applied Science) with GS Junior Sequencing Instruments; to recognize each specific patient, primer pairs included a 10-base molecular identifier barcode sequence (MID). We generated amplicon ultra-deep sequencing of the 2 amplicons with a median coverage per amplicon of 1800 reads per sample. Twenty patients (55,6%) out of 36 with t(4;11) at diagnosis harbored KRAS mutations (range of penetrance of 0,4–29,7% for mutated sequences). Interestingly, in some patients more than one clone with different mutations at difference penetrance in KRAS codons were present. Of the 18 relapse samples 4 (22%) harbor KRAS mutations (penetrance of 1.3–38% of reads). Sanger sequencing was used to validate the presence of mutations in patients with more of 20% of penetrance identified with 454 technology. To evaluate the role of KRAS mutations in the leukemogenic process we analyzed matched paired diagnostic and relapse samples; in patients with mutations at diagnosis, 3 distinct situations were identified in the relapsed samples, pointing different changes between diagnosis and relapse: (1) presence of KRAS mutations in a specific codon at diagnosis and disappearance of this clone at relapse (6 of 9 cases, 67%); (2) decrease of the penetrance of a mutation between diagnosis and relapse and appearance of a new different mutation at relapse (2 of 9 cases, 22%); (3) clonal evolution of the same mutation and increase of the percentage of the mutated sequence from diagnosis to relapse (1 of 9 cases, 11%). Apparently, patients with MLL-AF4 rearrangements at diagnosis and relapse presented with subclones and during the relapse phase previous clones disappeared while new clones appeared and only in one case we can assume a selective advantage of the clone with the KRAS mutation. To further explore the propagation of KRAS mutated subclones in MLL-AF4, we transplanted 2 patients at diagnosis into a NOD/SCID xenograft mouse model. Xenografted samples obtained from leukemia bearing mice transplanted further onto subsequent recipients in serial passages. KRAS mutations were analyzed for each of the passages as mentioned above. One of the diagnostic samples used for transplantation harbored KRAS G12S mutation. The human cells of this patient isolated after the first passage in the mouse carried the same mutation of the diagnosis but with a very low penetration, suggesting a minority clone that decreased further in the second passage. No trace of mutations was found in the third passage. The other diagnostic sample for which serial transplanted samples were analyzed had no KRAS mutation at diagnosis and no new mutations appeared during further passages in the mice. The disappearance of the mutated KRAS subclone during passages in the recipient mice seems to reflect the negative selection for mutated clones observed in patients between diagnosis and relapse. Overall, these data showed that KRAS mutations are frequently occurring in MLL-AF4 leukemias at diagnosis however these mutations do not seem to add a selective advantage to the blast cells. Disclosures: No relevant conflicts of interest to declare.

Abstract 3907 Acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy. Although the vast majority of patients initially respond to up-front chemotherapy, relapses occur in approximately 20% of cases and have a poor prognosis. Thus, novel therapeutic strategies are required to treat minimal residual disease and improve long-term survival. B cell precursor (BCP)-ALL cells express low levels of costimulatory and antigen-presenting molecules and therefore are poorly recognized by the immune system. Previous reports show that CpG oligodeoxynucleotides (CpG) can induce immunogenicity of non-Hodgkin's lymphomas including B-CLL, and that CpG may decrease BCP-ALL tumor burden in a murine xenograft model. In the present study we investigated the effect of various combinations of known potent B cell stimulators including CpG, Interleukin (IL)-2 family cytokines and CD40 ligand (CD40L) on the immunogenicity of both primary BCP-ALL cells and a series of established BCP-ALL cell lines. Using combinations of CpG, IL-4 and CD40L we were able to enhance expression of CD40, CD54, CD86 and MHC class II on BCP-ALL cells, resulting in an increased capacity of BCP-ALL cells to induce proliferative T cell responses and to generate cytotoxic T cells (CTLs). Of note, these CTLs exhibited significantly enhanced anti-leukemic cytotoxicity not only towards treated but also towards untreated BCP-ALL cells, with similar effects found in both allogeneic and syngeneic settings. The strongest effects were observed after exposure of BCP-ALL cells to a combination of CpG, IL-4 and CD40L, whereas untreated control BCP-ALL cells induced minimal T-cell proliferation and cytotoxicity only. Importantly, CpG in combination with IL-4 and CD40L was significantly more effective than CpG alone. Our results demonstrate that combined treatment with CpG, IL-2 family cytokines and CD40L is more efficient than CpG alone in inducing an immunogenic phenotype in BCP-ALL cells. Further studies using a humanized leukemia mouse model may open novel therapeutic approaches in the management of BCP-ALL, for example by using adoptive T cell transfer after in-vitro stimulation of autologous T cells with activated BCP-ALL cells. Disclosures: No relevant conflicts of interest to declare.

Abstract 2722 Defects in cell death signaling might be responsible for treatment failure and development of relapse in acute leukemia. In two retrospective studies we recently investigated the functional integrity of apoptosis signaling in primary patient leukemia samples and reported an importance of intact apoptosis signaling for good outcome in pediatric ALL and AML. Using a NOD/SCID/huALL mouse xenotransplant model for pediatric ALL we have also shown that rapid engraftment of primary ALL cells (short time to leukemia/TTLshort) is characteristic for early relapse of the corresponding patient. The aim of this prospective study was to analyze the impact of the functional integrity of apoptosis signaling in xenograft ALL samples on NOD/SCID engraftment and patient outcome. Furthermore, expression of apoptosis regulating molecules was investigated and correlated to NOD/SCID engraftment, response to treatment and apoptosis signaling. Primary BCP-ALL samples (N=20) obtained at diagnosis were transplanted onto NOD/SCID mice and time to leukemia (TTL) was estimated as weeks from transplant to onset of disease for each sample transplanted. Apoptosis signaling was investigated in xenograft leukemia samples analyzing two key apoptogenic events by flowcytometry, cytochrome c release and caspase-3 activation. Transcript levels of the anti-apoptotic molecules Mcl-1, XIAP, Bcl-2 and Livin was analyzed by quantitative RT-PCR. Of the 20 ALL samples transplanted 6 led to rapid leukemia manifestation in the recipient animals (TTLshort) with an inferior relapse free survival of the corresponding patients in contrast to 14 patients with TTLlong phenotype (log rank P.002). Apoptosis signaling was investigated in xenograft leukemia samples by analysis of caspase-3 activation and cytochrome c release. Both events were closely correlated to each other indicating intact signaling in xenograft leukemia derived from patients stratified into non-high risk groups, patients showing good response to treatment (remission on day 15, negative MRD on day 33), and patients without relapse. In contrast, no correlation was found in xenografts from patients with poor outcome. Most importantly, intact apoptosis signaling was also found in TTLlong but not in TTLshort patients strongly indicating that mitochondrial cytochrome c release and consecutive apoptosome formation resulting in activation of downstream effector caspases such as caspase-3 is characteristic for a long engraftment phenotype and prognostic favorable ALL. The functional integrity of this apoptogenic checkpoint is subsumed by the parameter cytochrome c related activation of caspases (CRAC). Patients with positive CRAC values reflecting intact apoptosis signaling showed a significantly superior relapse free survival in contrast to patients with disturbed cytochrome c related caspase activation/negative CRAC (log rank, P Transcript expression of apoptosis regulating molecules was analyzed. A correlation of cytochrome c release and caspase-3 activation indicating proficient apoptosis signaling was exclusively observed in leukemia samples with low expression of the anti-apoptotic molecules Mcl-1, XIAP, Bcl-2 and Livin. Furthermore, patients samples with low Mcl-1 expression showed a significantly longer time to leukemia (TTL) and lower blast cells on day 8 than samples with high Mcl-1 expression. In summary, the propensity of leukemia cells to undergo apoptosis (CRAC positive) leads to prolonged engraftment upon transplant in the NOD/SCID/huALL model (TTLlong), is associated with low expression of anti-apoptotic molecules, and results in favorable treatment response and superior survival of pediatric ALL patients. Disclosures: No relevant conflicts of interest to declare.

Abstract 2653 Poster Board II-629 Oncogenesis and tumor progression are supported by alterations in cellular signaling. We used phospho-specific antibodies in combination with surface staining in flow cytometry to analyze specific signaling profiles of leukemia cells at a single cell level. We anayzed 22 xenograft samples derived from NOD/SCID-mice transplanted with primary pediatric B- cell precursor acute lymphoblastic leukemia (BCP- ALL) cells. The cells were isolated from the spleens of leukemia bearing mice and stimulated ex vivo in vitro with different stimulants and cytokines. Activation of various phosphoepitopes was analyzed by flow cytometry and compared to the basal state of unstimulated samples. TEL/AML1 fusion and MLL-rearrangements are the most common cytogenetic aberrations in childhood BCP- ALL and are associated with a good or very poor prognosis, respectively. Although there were no differences detectable in basal phosphorylation between the different cytogenetic subgroups, TEL/AML1- positive samples (n= 5) displayed a significantly lower phosphorylation of extracellular regulated kinase (ERK1/2) after stimulation with PMA (Phorbol-12-myristat-13-acetate, activator of protein kinase C) or interleukin 7 (IL-7), while they showed a significantly higher activation of p38 after stimulation with PMA, compared to samples without translocation (n= 13). Additionally, the fusion gene negative samples showed a downregulation of STAT1-phosphorylation after stimulation with interleukin 10 (IL-10) whereas the TEL/AML1-positive samples showed no change. Interestingly, the MLL- positive samples (n= 3) also did not show a difference in STAT1-phosphorylation after IL-10, but showed significantly stronger STAT1 activation in response to interferon alpha (IFN-a) compared to samples without fusion genes. Moreover, the MLL- positive samples also displayed a weaker reaction in ERK-phosphorylation after IL-7 compared to the leukemia samples without cytogenetic aberrations. Differences in other prognostic subgroups analysed include a weaker phosphorylation of p38 and JNK after anisomycin in samples where the patient initially presented with hyperleucocytosis (> 100.000 WBC/μl) (n= 3), an indicator of poor prognosis. A decrease in STAT3- activation after IL-10 was observed in samples where the patients displayed bone marrow remission on day 15 of therapy (n= 8), compared to no change in the samples of patients with > 5% residual blasts (n= 8), indicative of therapy resistance, at this timepoint. Similar to the results for the cytogenetic subgroups, there were no differences detectable at basal phosphorylation levels between the prognostic subgroups. Taken together, these data show that basal phosphorylation states of specific signaling molecules do not discriminate between the different prognostic subgroups of BCP- ALL analyzed. Cytogenetic and other prognostic subgroups however display specific profiles of signaling networks after stimulation. This strategy will prove helpful to identify mechanisms by which different subgroups with distinct clinical outcomes interpret environmental signals and hereby define pathways important for continued survival, proliferation and resistance eventually leading to novel biomarkers and targeted therapies. Disclosures: No relevant conflicts of interest to declare.

Abstract 1630 Poster Board I-656 Acute lymphoblastic leukemia (ALL) is the most frequent malignant disease in childhood. Despite efforts achieved in treatment resulting in improved long term survival about 20% of the patients encounter relapse of their disease. Involvement of the central nervous system (CNS) is a common feature at diagnosis but also at relapse. About 10% to 20% of relapse patients present with isolated CNS leukemia as first event mainly occurring early within 24 months after diagnosis and associated with a survival of about 50%. However, achievement of effective CNS control is essential for successful treatment. Model systems to study the mechanisms of CNS leukemia but also new treatment strategies are required. Here we present and characterize a new pediatric B cell precursor (BCP) ALL cell line (018Z) which was established by passage in a NOD/SCID/huALL xenotransplantation model and exhibits a distinct CNS phenotype if re-transplanted onto NOD/SCID mice. Leukemia cells isolated from a male, adolescent patient at diagnosis were transplanted onto unconditioned NOD/SCID mice by intravenous injection. The patient had no CNS disease at diagnosis, however he died in remission after induction therapy. Upon onset of leukemia in the recipients the animals were killed and the engrafted leukemia cells were isolated and further transplanted onto secondary and tertiary recipients. Cells isolated from subsequent recipients were transferred into cell culture flasks and kept under standard culture conditions in RPMI medium supplemented with 20% fetal calf serum. Autonomous proliferation of the cells was observed for now more than three years. The cell line remained immunophenotypically stable as regularly assessed by flowcytometry (every 20 passages, from cell line passage 20 up to 160) showing the same BCP-ALL subtype. Infection of the cells with Epstein Barr Virus or Human Herpes Virus 8 was ruled out. Cytogenetic analysis of the cell line revealed presence of 47, XY, +8, del(9)(p13). The same karyotype was found in two samples isolated from previous xenograft passages (P4 and P6) and in leukemia cells obtained at diagnosis. The same karyotype was also detected if the 018Z cell line cells were re-transplanted and leukemia cells isolated from the recipients. Interestingly, at diagnosis two additional karyotypes were observed but could not be detected in the xenografts or cell line indicating selection and subsequent proliferation of one clone engrafted [47, XY, +8, del(9)(p13)]. Interestingly, chromosome arm 9p abnormalities have been reported to be associated with adverse events in pediatric BCP ALL, including onset of CNS leukemia (Heerema et al., Blood, 1996, 94(5): 1537-44). The cell line was further characterized and re-transplanted in the NOD/SCIDhuALL model. Unlike leukemia manifestation observed after transplantation of other samples, recipients transplanted with the 018Z cell line presented with a rapid onset of symptoms like seizures and palsies of the limbs pointing to CNS involvement. Infiltration of BCP ALL cells in the CNS of the recipients was detected by immunohistochemistry of the brain and by flowcytometry in cell suspensions prepared from the mice' meninges while only below 5% of human leukemia cells were detected in peripheral blood, spleen or bone marrow. Taken together, we established a novel BCP ALL cell line showing manifestation of CNS leukemia with rapid onset of about 6 weeks after re-transplantation onto NOD/SCID mice and providing a model system to study CNS disease in ALL. Disclosures No relevant conflicts of interest to declare.

We previously identified the importance of intact apoptosis signaling for treatment response in primary pediatric ALL and AML by analysing two key apoptogenic events, caspase-3 activation and cytochrome c release. A correlation of both events was exclusively found in the groups of patients with good response to treatment, showing that intact apoptosis signaling is a critical feature for favorable outcome. Using a NOD/SCID mouse model for pediatric B cell precursor ALL we found that rapid in vivo leukemia growth (Time to leukemia (TTL) short) in transplanted recipients determines poor patient outcome. In this study we now investigated the importance of deficient apoptosis signaling for leukemia engraftment in this model. We analyzed a total of 17 BCP-ALL xenografts, 5 of which had a short time (TTLshort) to clinical manifestation of the disease (< 10 weeks) and 12 with a significantly longer time (TTLlong) to leukemia (> 10 weeks). The apoptosis signaling parameters cytochrome c release and caspase-3 activation were evaluated flowcytometrically in directly isolated cells from the leukemia bearing mice before and after induction of apoptosis by factor deprivation in culture and related to TTL. Comparing TTLshort and TTLlong a significant correlation of both apoptosis parameters was found in the TTLlong group only (P= .017). Importantly, patients whose blasts engrafted in NOD/SCID-mice with TTLlong showed a superior outcome compared to the TTLshort group. Additionally, proficient apoptosis signaling as indicated by the correlation of cytochrome c release and caspase-3 activation, was only found in blast cells from recipients where the corresponding patient had favorable prognostic factors such as TEL/AML1-positivity (P= .023), absence of hyperleucocytosis at diagnosis (P= .012), absence of relapse (P= .038) and no early relapse (P= .017). In contrast, the correlation of cytochrome c release and caspase-3 activation was absent in the groups with poor prognosis. We also found a direct positive correlation between TTL and cytochrome c (P= .022) and TTLlong and caspase-3 activation (P= .048), indicating that a longer engraftment time may be due to proficient apoptosis signaling in the leukemic blasts. Our finding in the NOD/SCID human-ALL model matches our retrospective results in pediatric ALL and AML to conclude that the functional integrity of a downstream apoptotic checkpoint (cytochrome-c related caspase activation) is an important feature regulating leukemia biology. Thus, deficient apoptosis signaling appears to determine rapid engraftment of leukemia cells in the NOD/SCID model in vivo and consequently poor patient outcome.

Oncogenesis and tumor progression are supported by alterations in cellular signaling. We used phospho-specific antibodies in flow cytometry to analyze specific signaling profiles of leukemia cells at a single cell level in 7 B cell precursor (BCP)-ALL leukemia cell lines and 7 primary pediatric BCP-ALL xenograft samples. Peripheral blood lymphocytes gated on CD19-positive B cells were used as normal nonmalignant controls. Cells were stimulated by different stimulants and cytokines (PMA, anisomycin, IL-4, IL-6, IL-7, IL-10 and IFN-α) and activation of various phosphoepitopes (pERK, pp38, pJNK, pStat1, pStat3, pStat5, pStat6) was analyzed and compared to the basal state of unstimulated samples. Signaling profiles of normal B-lymphocytes were compared to those of the BCPALL cell lines as well as to the BCP-ALL xenograft samples. Significance of differences was assessed by the nonparametric Mann-Whitney U-test. Basal phosphorylation was significantly higher in the leukemia cell lines than in normal lymphocytes. Similarly, basal phosphorylation of all analyzed epitopes in xenografts exceeded the phosphorylation state of normal B-lymphocytes (with the exception of p38 phosphorylation, where there was no significant difference). Interestingly, the BCP-leukemia cell lines also had significantly higher basal phosphorylation levels than the primary BCP-ALL xenografts. However, when comparing the amounts of phosphorylation before and after stimulation mature normal B-cells displayed significantly higher profiles compared to the leukemia cell lines e.g. for pp38 and pJNK after stimulation with PMA (P= .001), for pStat3 after stimulation with IL-6 (P= .002) and IL-10 (P= .037) and for pStat6 (P= .001) after stimulation with IL-4. Conversely, the leukemia cell lines showed increased phosphorylation of p38 after stimulation with anisomycin (P= .021) as well as higher Stat5 phosphorylation after stimulation with IL-7 (P= .021) compared to normal lymphocytes. In normal B-cells compared to xenografts higher levels were found after stimulation with PMA for pp38 (P= .007), for pJNK after PMA stimulation (P= .001), for pStat3 after IL-6 (P=.003) and for pStat6 after IL-4 (P= .002) stimulation while the xenograft samples displayed stronger reaction to stimulation with anisomycin for pp38 (P= .037) and to stimulation with IL-7 for pStat5 (P= .028). The level of phosphorylation after treatment with different stimulants in the xenografted leukemia samples was similar to that of the leukemia cell lines although the cell lines displayed higher basal phosphorylation values. The BCP-leukemia cell lines and the BCP xenograft samples both displayed high levels of constitutive phosphorylation in general reducing their ability to react to a given stimulus compared to normal B-lymphocytes. With the most important exception of Stat5: we consistently found that Stat5 phosphorylation is increased in acute lymphoblastic leukemia cell lines and primary xenografts after stimulation with IL-7 compared to normal B-lymphocytes. Stat5 is known to enhance proliferation and protect from apoptosis and our data now strongly suggest that Stat5 and Stat5 dependent pathways are critically involved in leukemogenesis. Since we could identify significant and specific phosphorylation signatures characteristic for leukemia cells, this provides a strategy to define pathways important for continued survival, proliferation and resistance of leukemia and allows identification of therapeutic targets and novel biomarkers associated with clinical outcome.

Acute lymphoblastic leukemia (ALL) is the most frequent malignant disease in childhood. Advances in therapy, in particular stratification of patients to appropriate treatment according to risk factors improved long term survival attaining cure rates of up to 80 %. Despite the efforts achieved by the stratification strategies the majority of relapse patients are recruited from the low risk groups emphasizing the need for additional independent risk factors. In a recent study we transplanted primary leukemia samples obtained from pediatric patients with newly diagnosed B cell precursor ALL (BCP-ALL) into NOD/SCID mice. Time to leukemia (TTL) was analyzed for each patient sample transplanted from date of transplant to date of leukemia manifestation in the recipients. We demonstrated that patients whose leukemia cells engrafted rapidly leading to manifestation of the disease within 10 weeks (TTLshort) showed a clearly inferior relapse free survival in contrast to patient samples with prolonged in vivo growth (TTLlong). Interestingly, the same distinct difference in relapse free survival was observed in the low risk groups only. Multivariate analysis showed an almost 45- fold increased risk for relapse in TTLshort patients. In order to further characterize the biological properties of the leukemia cells in the two groups, gene expression profiles of samples with short versus long TTL in the xenograft model were analyzed using a human whole genome array (Affymetrix U133 Plus 2.0). Here, we used quantitative traits analysis (QTA) correlating gene expression values (relative expression) to the time from transplant to manifestation of leukemia in the NOD/SCID mice (TTL, in weeks). 14 different xenograft samples (TTLshort n= 7, mean TTL 8.14 weeks; TTLlong n= 7, mean TTL 19.71 weeks; T-test P = .03) isolated from leukemia bearing mice were investigated. All 14 patients included were stratified in low (standard or intermediate) risk groups. All patients were negative for TEL/AML1- fusion, BCR/ABL- fusion or MLL rearrangement. By QTA we identified 5 genes that were significantly correlated (Spearman correlation, for all 5 genes P < .0001) to time from transplant to leukemia manifestation in the recipient mice (TTL). Analysis of the 14 xenograft samples using the 5 genes identified showed clustering of all but one sample in one group. The 5 genes were than explored for their power to predict relapse in an independent cohort of pediatric ALL patients. For these patients expression profiles were analyzed in leukemia samples obtained at diagnosis. All patients in this cohort were also stratified in low risk groups and negative for the presence of TEL/AML1-, BCR/ABL- or MLL- fusion transcripts. 8 of the 38 patients encountered relapse, 5 at early and 3 at late time points (< or > 24 months after diagnosis). Clustering according to the 5 genes identified by QTA lead to separation of the patients into two groups. Interestingly, all relapsed patients except one clustered in the group associated with the signature characteristic for TTLshort. Most importantly, all 5 patients with early relapse gathered in this cluster. Taken together, we used a novel approach directly correlating gene expression values to the continuous variable time to leukemia (TTL) which might be reflected more accurately by this strategy than comparing two groups. We applied this gene signature characteristic for TTLshort, thereby identifying poor overall survival in a set of independent pediatric ALL patients and found clustering of all early relapse patients in one group. This new independent risk factor might identify patients with high risk for early relapse avoiding xenografting in the mouse model.

Drug resistance and treatment failure in acute leukemia may be attributed to apoptosis resistance in leukemia cells since defects in apoptosis signal transduction are commonly acquired during malignant transformation. Expression analysis of single molecules with regard to clinical outcome could so far not identify common apoptosis defects with prognostic value in primary acute leukemias. We addressed the role of apoptosis signaling for the initial response to induction treatment in pediatric B precursor lymphoblastic leukemia by functional assays. Apoptosis signaling in response to survival factor withdrawal was assessed in 82 primary leukemia samples by measurement of the key apoptotic events, caspase-3 activation and mitochondrial cytochrome c release. Caspase-3 activation directly correlated to the initial response to treatment assessed by the percentage of leukemia cells in bone marrow on day 15 (p = 0.015). Intact apoptosis signaling was indicated by a significant correlation of caspase activation and cytochrome c release was found especially in the groups of good responding patients (rs 0.375 – 0.502; p = 0.001). Interestingly, this correlation was completely absent in all groups of poor responding patients defined by insufficient leukemia cell reduction on day 8, 15 or 33. The efficacy of apoptosis signal induction in the individual leukemia is expressed by the parameter CRAC (Cytochrome c Related Activation of Caspase-3), relating the extent of caspase activation to cytochrome c release in a single patient sample. Ten of twelve patients with prednisone poor response (chi square, p= 0.031) and all patients not achieving remission on day 33 (4 of 4) had negative CRAC values, indicating deficient cytochrome c related caspase activation. Furthermore, the CRAC negative group revealed significant higher percentages of leukemia cells in bone marrow on day 15 (n=37, mean 22.8) than the CRAC positive patients (n=30, mean 6.2, p = 0.004). In addition all four relapse patients showed negative CRAC values indicating a prognostic value beyond initial treatment response. The data show that the intact relation of cytochrome c release and caspase activation indicating an intact apoptosome function is important for efficient remission induction treatment in childhood B precursor ALL. Assessment and quantification of this relation for individual patients by the parameter CRAC may serve as potential factor for treatment stratification.

In acute lymphoblastic leukemia (ALL), central nervous system (CNS) directed therapy is required to achieve long-term remission and survival even in patients without detectable CNS disease, indicating subclinical CNS manifestation in many patients. Hence, prophylactic CNS therapy is indispensable but bears the risk of secondary neoplasms and neurocognitive deficits in ALL survivors. Therefore, a better understanding of mechanisms mediating CNS involvement in ALL are needed in order to identify potential targets for prophylactic and therapeutic intervention. In this study, we transplanted primary patient B cell precursor (BCP) ALL cells onto NOD/SCID mice and investigated engraftment of human leukemia cells in the recipient's peripheral blood (PB), bone marrow (BM), spleen (S), and meninges by flowcytometry staining for huCD19. Upon disease onset, we identified meningeal infiltration of human ALL cells together with leukemia engraftment in BM, S and PB in a subset of samples (CNSpos) in contrast to absent CNS involvement despite high leukemia cell infiltration of BM, S and PB in other recipients (CNSneg). CNSpos and CNSneg phenotypes were consistently observed in subsequent xenograft passages. In CNSpos animals meningeal leukemia infiltration was also detected by immunohistochemistry on brain sections and meningeal enhancement was detected by small animal magnetic resonance imaging in CNSpos recipients. We further characterized ALL cells isolated from meningeal and BM infiltrates by gene expression profiling and identified the gene coding for vascular endothelial growth factor A (VEGF) to be highly expressed in ALL cells isolated from the CNS as compared to BM derived cells. Differential expression of VEGF was validated by qPCR and confirmed in independent sample cohorts. Most interestingly, reported functions of VEGF include regulation of cellular growth, vascular permeability, and trans-endothelial cell migration, and elevated VEGF protein levels have previously been described in cerebrospinal fluid specimens collected from ALL and AML patients with CNS leukemia. VEGF signals through its receptors 1 or 2 (VEGFR1/2). On all primary ALL samples, only expression of VEGFR1 but not VEGFR2 was detected. We used the BCP-ALL cell line Nalm-6, which also expresses VEGF and VEGFR1 and mediates a clear CNSpos phenotype upon engraftment. However, cellular survival, proliferation, apoptosis, and metabolic activity were not affected, neither upon exposure to VEGF or the antagonizing VEGF antibody bavacizumab, nor by stable VEGF overexpression or knock-down, thus indicating absence of autocrine VEGF/VEGFR1 signaling. We further analyzed whether paracrine VEGF signaling through VEGFR2, which upon activation mediates endothelial cell permeability, is involved in trans-endothelial migration of ALL cells leading to leukemia infiltration of the CNS. Brain endothelial cells (bEND.3) incubated with VEGF showed increased phosphorylation of VEGFR2 downstream signaling molecules (Src, AKT), which indicates activated signaling mediating cellular permeability. Moreover, we modeled migration of ALL cells through brain endothelial cells in a transwell assay and observed significantly increased migration of Nalm-6 ALL cells through bEND.3 monolayers upon exposure to VEGF or upon overexpression of VEGF as compared to controls. Vice versa, significantly lower numbers of migrated leukemia cells were detected after incubation with bevacizumab or upon VEGF knock down indicating VEGF dependent trans-endothelial migration. Thus, we identified high expression of VEGF in CNS derived leukemia cells, absent autocrine signaling of VEGF on ALL cells and, most importantly, VEGF dependent trans-endothelial migration of ALL cells indicating VEGF as a possible mediator of CNS leukemia. Finally, we investigated the impact of VEGF on CNS leukemia manifestation in vivo. Recipient animals transplanted with 3 CNSpos primograft samples (4 experiments, one in repetition) were treated with bevacizumab or control. In all experiments, anti-VEGF treatment significantly reduced the leukemia burden exclusively in the CNS but not in BM, S, and PB compartments, indicating that transmigration of leukemia cells and CNS manifestation in ALL is mediated by VEGF. Thus, targeting of VEGF signaling may serve as a novel strategy to control CNS leukemia in patients. Disclosures No relevant conflicts of interest to declare.