BCP-ALL Cell Lines Research Articles

The Tetraspanin CD9 Shapes Glucocorticoid Sensitivity in Pediatric B-Precursor Acute Lymphoblastic Leukemia

Background: De novo or acquired resistance to glucocorticoids (GCs), the common therapeutic regimens for remission-induction in pediatric B-precursor acute lymphoblastic leukemia, confronts treatment success and is a strong predictor for disease relapse. Discovery of predictive markers and underlying mechanisms of GC resistance could lead to new treatment and prognostic modalities. Aims: This study aims: (1) to demonstrate the association between CD9 expression and GC sensitivity in childhood BCP-ALL; (2) to dissect the underlying mechanisms driving GC resistance; and (3) to discover the possible therapeutic modalities for restoring GC susceptibility. Methods: Ex vivo drug sensitivity profiling to prednisone (Pred) and dexamethasone (Dex) was performed on 6 BCP-ALL cell lines and 18 patient samples with differential CD9 expression. Association of CD9 expression with patient responses to Pred prophase was analyzed in a pediatric BCP-ALL cohort (n=182). Functional impact of CD9 on GC susceptibility was dissected by gain- and loss-of-function experiments in BCP-ALL cells. Mechanistic basis of CD9-driven GC response was investigated through genomics, transcriptomics, and proteomics. Reversal of GC resistance by pathway-targeted agents were explored on BCP-ALL cells and xenograft models. Results: Distinctive differences in the drug sensitivity pattern were found between CD9- and CD9+ BCP-ALL cell lines, with the former showing resistance to GCs but not other chemotherapeutic agents. Concurrently, CD9- primary lymphoblasts exhibited resistance to Dex (IC50s: 2,761 vs. 31 nM) and Pred (IC50s: 8,185 vs. 268 nM) compared with CD9+ samples (positivity defined by the presence of ≥ 20% CD9+ blasts). More Pred poor responders (defined by the presence of ≥ 1×10 9/L circulating lymphoblasts on day 8) were found in CD9- than CD9+ patients (19.4% vs. 6.2%, P=0.018). Subgroup analyses revealed more poor responders with CD9- phenotype were enriched in patients with older age (60% vs. 3.7%, P=0.008), male gender (21.7% vs. 6.7%, P=0.045), higher white blood cell count (66.7% vs. 15.4%, P=0.004), and in those with BCR-ABL1 translocation (100% vs. 12.5%, P=0.024), or not otherwise cytogenetically specified BCP-ALL (30.8% vs. 5.8%, P=0.019). Univariate and multivariate analyses further confirmed CD9 negativity as an independent predictor for GC resistance (OR=5.1, P=0.009). Overexpression of CD9 in CD9- cells (SEM, KOPN-8) by lentiviral transduction significantly enhanced GC sensitivity by 2.8-62.3-fold ( P<0.024), whereas CD9 knockout in CD9+ cells (697) using CRISPR-Cas9 decreased GC susceptibility ( P<0.003). No significant differences in mRNA or protein expression of the GC receptor NR3C1 were identified between CD9+ and CD9- cells, as well as its isoforms or mutants. The cytoplasm-to-nucleus translocation of GC receptor was consistent among cell lines regardless of CD9 expression status, but CD9 was physically interacted with NR3C1. Upon GC stimulation, the presence of CD9 increased the number of DEGs (110 vs. 82 DEGs) and magnitude of GC-responsive genes exclusively detected in the background of CD9 (1.3-18.6-fold; e.g. AKAP13, STAT5A, EZR). CD9- cells showed constitutive activation of the MAPK pathway and could be preferentially restored for GC susceptibility by the MEK1/2 inhibitor trametinib in vitro. BCP-ALL xenograft modeling revealed that, compared to single agent Dex treatment, combination with trametinib significantly reduced the leukemic load in animal transplanted with CD9- SEM but not CD9+ BV-173 cell lines. Ex vivo drug testing further showed that the resistance to GCs in some CD9+ cases could be partially reversed by combination of trametinib and the JAK inhibitor ruxolitinib due to the activation of STAT5. Conclusions: This study provides a definitive linkage of CD9 with GC sensitivity in childhood BCP-ALL mediated through a NR3C1-independent mechanism. CD9- cases could be prone to trametinib combination therapy, whereas CD9+ cases might need a third drug such as ruxolitinib to counteract GC resistance.

TP53 Aberrant B-Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL) Are Uniquely Sensitive to a Combination of Pyrimidine De Novo Synthesis Blockade and Replication Checkpoint Inhibition

Background Unlike most solid tumors, the incidence of TP53 aberrations in ALL is only 3% at diagnosis. However, in relapsed ALL this number increases to 12%. More importantly, at relapse, loss of p53 function is associated with therapy failure, with an overall survival of only 20%. Hence, there is a clear clinical need for more effective strategies to treat TP53 aberrant relapsed ALL. Owing to their high proliferative state, leukemic blasts show a high dependency on pyrimidines for DNA synthesis. A lack of pyrimidines leads to replication stress, causing activation of p53 and the DNA damage response pathway. Results We used CRISPR/Cas9 to generate TP53 deficient variants of the TP53 proficient BCP-ALL cell lines Nalm6 and RCH-ACV. We used these cell lines in a CRISPR screens and high throughput drug screen, and identified a strong synergy between the targeting of pyrimidine synthesis and DNA checkpoint kinase ATR and its downstream effectors Chk1 and Wee1 in TP53 deficient ALL. To validate these results, we tested the efficacy of a checkpoint inhibition together with Orludodstat (BAY-2402234), an inhibitor of Dihydroorotate Dehydrogenase (DHODH), a key enzyme in de novo pyrimidine biosynthesis. This clinical grade inhibitor was evaluated in either wild type or TP53 deficient BCP-ALL and T-ALL cell line models and patient derived xenografts (PDX). A strong apoptotic response was observed when Orludodstat was combined with the ATR inhibitor Berzosertib, most notably in TP53 deficient ALL. This p53 dependency was confirmed using ex vivo cultures of patient derived xenografts (PDX) of diagnosis and relapse pairs, where each diagnostic sample was wild type for TP53, and the relapse carried a TP53 aberration. While little or no synergy was observed in the diagnostic samples, a strong efficacy of the combination was observed in the matched TP53 deficient relapsed samples (Figure 1 A). Preliminary results from i n vivo experiments using p53 deficient patient derived xenografts indicate that the combination of Orludodstat and Berzosertib is well tolerated, while significantly delaying leukemic growth (Figure 1 B). Conclusion Together, our findings suggest that the combined targeting of de novo pyrimidine synthesis and ATR signaling may present a specific and non-genotoxic vulnerability for TP53 aberrant ALL.

Restored Biosynthetic Pathways Induced By MSCs Serve As Rescue Mechanism in Leukemia Cells after L-Asparaginase Therapy

The cause of relapse in childhood acute lymphoblastic leukemia (ALL) is often associated with resistance to the standard chemotherapy treatment. The primary goal of this project was to elucidate the resistance mechanism of L-asparaginase (ASNase), one of the crucial drugs used in ALL therapy. The cytotoxic effect of ASNase relies on the depletion of extracellular asparagine (Asn) and glutamine, which is disastrous for leukemic cells since they have minimal activity of de novo synthesis of these amino acids that paradoxically have essential roles in leukemic cells' metabolism. We previously showed that ASNase caused metabolic reprogramming by which leukemic cells escaped the cytostatic effect of the treatment. In the present study, we investigated the role of the main aspects of the in vivo environment on the resistance mechanism of leukemic cells (BCP-ALL cell lines: NALM-6, REH, RS4-11 and SUP-B15 and primary ALL cells). By co-culturing them with mesenchymal stem cells (MSCs) and treating them with ASNase-pretreated culture media, we mimicked the bone marrow matrix and the in vivo half-life of the drug (1.28±0.35 days). The ASNase concentrations used in the ASNase-pretreated culture media were 0.04, 0.4 and 4 IU/mL. In concordance with previous results, we showed that leukemic cell survival was increased in the co-culture model compared to the "classical" in vitro treatment after five days using flow cytometry (NALM-6 - 0.04IU/mL: 17.37±2.8% p<0.0001, 0.4IU/mL: 18±2.8% p<0.0001, 4IU/mL: 25.87±2.3% p<0.0001; REH - 0.04IU/mL: 27.51±3.3% p<0.0001, 0.4IU/mL: 22.06±3.3% p<0.0001, 4IU/mL: 23.96±3.3% p<0.0001; RS4:11 - 0.04IU/mL: 50.07±2.9% p<0.0001, 0.4UI/mL: 60.14±2.9% p<0.0001, 4IU/mL: 34.50±3% p<0.0001; SUP-B15 - 0.04IU/mL: 13.90±2% p<0.0001, 0.4UI/mL: 19.55±2.3% p<0.0001, 4IU/mL: 5.72±2% p<0.05). While ASNase-mediated metabolic rewiring of leukemic cells persisted in both mono and co-culture: reduced glycolysis and increased fatty acid oxidation, the activity of mTOR-regulated biosynthetic pathways differed. The latter pathway was assessed by western blot quantification of the downstream targets of mTOR, S6 and CAD, which are protein and nucleotide synthesis mediators, respectively. In both cultures, the phosphorylated forms of S6 and CAD were inhibited after ASNase treatment (4IU/mL). However, the effect was significantly less profound in the co-culture model (REH: p-S6 (1.826 log (2) fold change, p=0.0043); p-CAD (2.385 log (2) fold change, p=0.0152), NALM-6: p-S6 (1.380 log (2) fold change, p=0.0106); p-CAD (0.78 log (2) fold change, p=n.s)). Similar changes in phospo-S6 were observed in primary BCP-ALL cells isolated from pediatric patients treated with ASNase. As shown by stable isotope tracing, asparagine synthesized de novo and released from MSCs compensated for asparagine depletion (after ASNase administration) and induced resistance of leukemic cells. Asparagine was sufficient to restore protein and nucleotide synthesis and partially rescued the viability of leukemic cells. In conclusion, the presence of MSCs sustains biosynthetic pathways, making leukemic cells more accessible to bioenergetic rewiring, which may counteract ASNase cytotoxicity. These findings present a potential therapeutical target for resistant patients. (Supported by GAČR GA20-27132S and GAUK 1262120)

Synergistic Activity of BH3-Mimetics By Combined Targeting of Anti-Apoptotic Regulators in B-Cell Precursor ALL

In acute lymphoblastic leukemia (ALL), the most frequent malignancy in children and adolescents, deregulated cell death pathways contribute to leukemia development and therapy failure. Apoptosis (programmed cell death) is controlled at the mitochondrial level by different pro- and anti-apoptotic regulators. Molecules of the BCL-2 family are key in regulating intrinsic apoptosis signaling. Pro-apoptotic BH3-only proteins such as BIM and BID activate pro-death proteins like BAX and BAK leading to cell death, while anti-apoptotic BCL-2 family members including BCL-2, MCL-1 and BCL-XL sequester pro-apoptotic molecules, thereby preventing pro-death protein activation and apoptosis induction. Small molecule inhibitors have been developed, which bind to the anti-apoptotic molecules BCL-2, MCL-1 and BCL-XL leading to cell death induction. In particular inhibition of BCL-2, a key regulator of survival in B-cell malignancies including BCP-ALL, by the specific inhibitor venetoclax (VEN) has shown substantial, clinical anti-tumor activity. However, in BCP-ALL heterogeneous sensitivities for VEN have been described suggesting that other BCL-2 family members like MCL-1 and BCL-XL interfere with BCL-2 inhibition thereby counteracting VEN activity leading to poor treatment response.In this study, we compared the effects of inhibition of the anti-apoptotic BCL-2 family members BCL-2 (venetoclax, VEN), MCL-1 (S63845) and BCL-XL (A-1331852), investigated molecular mechanisms and determinants of inhibitor sensitivity, and addressed potential synergistic activity upon simultaneous blockage of BCL-2 together with MCL-1 or BCL-XL in BCP-ALL.First, we investigated the activity of the single inhibitors to induce cell death (positive staining for propidium iodide) in 7 BCP-ALL cell lines and a series of 27 BCP-ALL patient-derived xenograft samples (PDX). Titrating increasing concentrations, we estimated half-maximal effective concentrations (EC 50) for each inhibitor showing heterogenous responses of individual samples to the different inhibitors. Importantly, sensitivities to either inhibitor were not associated with leukemia characteristics including recurrent genetic alterations described in BCP-ALL. We also did not observe similar sensitivities to the inhibitors of individual samples, however some VEN insensitive samples showed sensitivity to MCL-1 and/or BCL-XL-inhibition, suggesting functional substitution of the anti-apoptotic regulators.Next, we investigated anti-apoptotic addictions of BCP-ALL cells to BCL-2 family proteins upon VEN treatment. Analyzing apoptosis signaling after exposure to VEN and BH3-peptides, we identified that ALL cells adapt their anti-apoptotic addiction to MCL-1 and BCL-XL as an escape strategy from VEN-induced cell death. Analyzing protein complexes by co-immunoprecipitation, we found that exposure of ALL cells to VEN rapidly led to reduced BIM/BCL-2 and compensatory increased BIM/MCL-1 complexes. Conversely, S63845 reduced BIM/MCL-1 complexes and led to increased BIM/BCL-2 binding. Importantly, both protein complexes could be effectively disrupted by combination treatment with VEN and S63845, which resulted in release of BIM and promotion of apoptosis signaling.Based on our mechanistic findings, we evaluated BH3-mimetic combinations for cell death induction using multi-dose matrix assays to calculate synergy metrics based on the Bliss independence model. Analyzing combination effects in seven BCP-ALL cell lines and four PDX samples, we identified positive mean synergy scores of VEN with S63845 and A-1331852 in all cases. However, the most synergistic area of the multi-dose-response matrices differed among different leukemias, indicating different extents of addictions and thereby synergies at different drug concentrations.Finally, combined BCL-2 and MCL-1 inhibition was evaluated in vivo in a PDX model of KMT2A-ENL positive pro-B ALL. Combination treatment of VEN with S63845 led to reduced leukemia loads in spleen, bone marrow and CNS as compared to single agent treatment.Taken together, we found heterogeneous responses of BCP-ALL samples to BH3-mimetics antagonizing BCL-2, MCL-1 and BCL-XL. The ability of leukemia cells to adapt their anti-apoptotic dependency from BCL-2 to MCL-1 or BCL-XL can be used as target for combination therapy, demonstrating synergistic activity in PDX samples ex vivo and in vivo. DisclosuresNo relevant conflicts of interest to declare.

Methionine Restriction As a Novel Therapeutic Strategy for MLL (KMT2A)-Rearranged Acute Lymphoblastic Leukemia

Background:MLL (KMT2A)-rearranged acute lymphoblastic leukemia (MLLr ALL) is a rare but aggressive subset that represents 5% of childhood ALL cases, and accounts for about 70% of infant leukemias. While overall survival in these young children is around 50%, after relapse, MLLr ALL becomes an almost incurable disease, highlighting the urgent clinical need for new strategies for this patient group. The histone methyl transferase function of the MLL fusion protein complex requires the methionine metabolite s-adenosylmethionine (SAM) as methyl donor, suggesting a selective sensitivity of MLL-r ALL for perturbations in methionine availability. Recent studies in solid tumor models suggest clinical utility of methionine restricted diets or oral administration of methionine depleting enzyme Methionine Gamma Lyase (MGL) to be safe and effective. Therefore, we explored the effect of methionine restriction (MR) as a potential, new therapy for MLLr ALL.Methods:We compared the effects of MR on metabolic activity and viability between MLLr and non-MLLr pre-BCP ALL cell lines using enzymatic depletion, small molecule inhibitors targeting methionine metabolism, and restrictive culture conditions. To identify intrinsic metabolic differences between MLLr and non-MLLr cells and explore how MR impinges on their metabolic state, we performed global metabolomics on MLLr SEM cells and non-MLLr NALM6 cells cultured with complete depletion of methionine. Additionally, we used RNA sequencing to assess the global effects of MR on gene expression, and a CRISPR/Cas9-based reverse genetic screen to identify sensitizers towards MR. Results were validated in vitro using targeted knockouts and small-molecule inhibitors, as well as in vivo using a 95% methionine restricted diet. Immunocompromised mice were engrafted with MLLr SEM cells and 7 days after transplantation, mice were randomized to control or 95% MR diet. Leukemia progression was monitored by flowcytometric detection of human lymphocytes in the blood.Results:We observed that depletion of methionine reduces metabolic activity in almost all BCP-ALL (B-ALL) cell lines, however, only in MLLr B-ALL cell lines was rapid apoptosis induced (Figure 1A). Global metabolic profiling revealed significant basal metabolic differences, of note being SAM, whose levels were approximately 5-fold higher in MLLr SEM cells compared to non-MLLr NALM6 cells. Consistent with this, addition of SAM completely rescued MLLr cell lines from methionine depletion induced apoptosis, an effect not observed in non-MLLr cells (Figure 1A). Metabolomic profiling also highlighted different salvage mechanisms at play in NALM6 cells, with the folate cycle and polyamine synthesis pathway being activated upon MR. Together, these results indicate that MLLr B-ALL cells are selectively sensitive to MR. In line with this, RNASeq data showed significant decreased expression of several known MLL fusion target genes such as PROM1, HOXA10, and MEIS1 in response to MR.To obtain further insight into the pathways involved in the response to MR and to identify potential therapeutic targets that further sensitize cells to MR, we performed a CRISPR/Cas9-based screen. This identified three members of the Bromodomain- and extra-terminal domain (BET) family as potential modifiers of the response to MR in SEM cells. Indeed, RNAseq analysis showed that Myc activity as a proxy of BRD4 function, was strongly suppressed by MR. Finally, preliminary results show the efficacy of dietary intervention alone on leukemia progression. We observe with 95% MR diet, significant delays on leukemic growth (Figure 1B). Moreover, the MR diet was well tolerated, as indicated by minimal weight loss after two months. Although further studies are needed, we anticipate that targeting epigenetic regulators or use of conventional therapies in combination with MR would further potentiate this effect.Conclusions:MLLr leukemic cells have an increased dependency on S-adenosylmethionine and therefore show increased vulnerability to methionine depletion. Limiting methionine availability, either by enzymatic methionine depletion or dietary restriction could provide a novel therapeutic option for this patient group, particularly when combined with other therapies. The availability of an FDA approved methionine-free formula facilitates rapid translation to clinical practice, particularly in infants. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

CBFA2T3 Enhances Transcriptional Activity of RUNX1: Implication in Leukemia

Pre-B acute lymphoblastic leukemia (BCP-ALL) is the most common type of pediatric cancer. Many expression-profiling studies have uncovered genes deregulated in BCP-ALL compared to normal pre-B cells. Yet, the roles of the deregulation of those genes in the onset and maintenance of BCP-ALL are still unclear. We were interested in two genes, CBFA2T3 (Core-Binding Factor, Runt Domain, Alpha Subunit 2; Translocated To, 3) and RUNX1 (Runt Related Transcription Factor 1), which are specifically overexpressed in BCP-ALL characterized by the presence of ETV6-RUNX1 fusion gene. CBFA2T3 is a hematopoietic corepressor. Alteration of its function is implicated in leukemia. RUNX1 is a transcription factor, whose activation or repression activity depends on cofactors. RUNX1 deregulation is also involved in many hematological disorders. Our goal was to explore the role of these two genes in BCP-ALL. We demonstrated the major role of CBFA2T3 in the control of RUNX1 activity.Based on our patients' data and public database, we not only confirmed that CBFA2T3 and RUNX1 transcripts were overexpressed in ETV6-RUNX1 BCP-ALL, but also found that their expression levels were positively correlated. Expression of EVT6-RUNX1 in Nalm6 cells, a BCP-ALL cell line that normally does not express the ETV6-RUNX1 fusion protein, showed an overexpression of CBFA2T3 and RUNX1 proteins, confirming the role of the ETV6-RUNX1 fusion protein as a priming event in CBFA2T3 and RUNX1 expression.To further determine the role of RUNX1 in the control of transcription in the context of BCP-ALL, we performed chromatin immunoprecipitation followed by sequencing (ChIP-Seq) with anti-RUNX1 in Nalm6 cells and in REH cells, a ETV6-RUNX1 BCP-ALL cell line. Notably, we observed several significant enrichments of RUNX1 binding at CBFA2T3 and RUNX1 promoters in both cell lines. Our work on the promoters of these two genes was carried out with luciferase reporter assay. The luciferase results demonstrated that RUNX1 controlled the transcription of CBFA2T3 and RUNX1 and showed that CBFA2T3 enhanced the transcriptional activity of RUNX1.As CBFA2T3 is neither a transcription factor, nor a DNA binding protein, we suspected that CBFA2T3 could directly modulate RUNX1 activity. Chromatin immunoprecipitation of RUNX1 coupled to mass spectrometry identified a putative interaction between CBFA2T3 and RUNX1 in BCP-ALL lymphoblasts. Using Proximity Ligation Assay, we next confirmed the protein interaction between CBFA2T3 and RUNX1, as well as the interaction between CBFA2T3 and the fusion protein ETV6-RUNX1 in two BCP-ALL cell lines and, more importantly, in BCP-ALL patients' cells. Finally, we identified the CBFA2T3 protein domain responsible for the interaction with RUNX1, thanks to coimmunoprecipitation assay with truncated CBFA2T3. Using this CBFA2T3 domain, we next performed luciferase assay and were able to inhibit the effect of full length CBFA2T3 on the transcriptional activity of RUNX1.To conclude, this work highlights the interaction between CBFA2T3 with RUNX1 and ETV6-RUNX1. We identified a regulatory loop between CBFA2T3 and RUNX1 that could explain their deregulation in patients. Our finding reveals a novel molecular mechanism of control of RUNX1 involving CBFA2T3 and RUNX1 itself. It may also open new perspectives to understand the mechanisms implicated in acute myeloid leukemia with AML1-MTG16 (RUNX1-CBFA2T3) fusion protein.This work is supported by Ligue Régionale contre le cancer, Région Bretagne, Rennes Métropole, French Research Ministry, the société française de lutte contre les cancers et les leucémies de l'enfant et de l'adolescent, the Fédération Enfants et Santé, the Société Française de Biochimie et Biologie Moléculaire, the Société Française d'Hématologie, Mrs. M-Dominique Blanc-Bert and the Marie Curie Actions of the European Union FP7 under REA grant agreement n°291851. DisclosuresNo relevant conflicts of interest to declare.

Close interaction with bone marrow mesenchymal stromal cells induces the development of cancer stem cell-like immunophenotype in B cell precursor acute lymphoblastic leukemia cells.

Minimal residual disease of leukemia may reside in the bone marrow (BM) microenvironment and escape the effects of chemotherapeutic agents. This study investigated interactions between B cell precursor (BCP)-acute lymphoblastic leukemia (ALL) cells and BM mesenchymal stromal cells (BM-MSCs) in vitro. Five BCP-ALL cell lines established from pediatric patients and primary samples from a BCP-ALL patient were examined by flow cytometry and immunocytochemistry for expression of specific cell surface markers and cell adhesion proteins. The cell lines developed chemoresistance to commonly used anti-leukemic agents through adhesion to MSC-TERT cells in long-term culture. The change in chemosensitivity after adhering to BM-MSCs was associated with the expression of CD34, CD133, P-glycoprotein and BCRP/ABCG2, and downregulation of CD38. Similar phenotypic changes were observed in primary samples obtained by marrow aspiration or biopsy from a BCP-ALL patient. BM-MSC-adhering leukemia cells also showed deceleration of cell proliferation and expressed proteins in the Cadherin and Integrin pathways. These results suggest that BCP-ALL cells residing in the BM microenvironment may acquire chemoresistance by altering their phenotype to resemble that of cancer stem cells. Our results indicate that cell adhesion could be potentially targeted to improve the chemosensitivity of residual BCP-ALL cells in the BM microenvironment.

Abstract 2541: MicroRNA-497~195 cluster suppresses acute lymphoblastic leukemia growth by targeting CCND3/CDK4 and inhibiting cell cycle progression

Abstract Acute lymphoblastic leukemia (ALL) is the most common malignancy of childhood. Relapse is associated with poor prognosis, requiring further investigation on high-risk leukemias. MicroRNAs (miRNAs) are involved in the regulation of physiological processes. Deregulated miRNAs have been described to play a role in the initiation and progression of leukemia. Here, we investigated miRNA expression profiles comparing leukemia samples of patients with good and poor outcome, also characterized by slow or rapid engraftment, respectively, upon transplantation into NOD/SCID mice. We found downregulation of the miR-497~195 cluster in B-cell precursor (BCP)-ALL patient-derived xenograft samples with rapid engraftment and early relapse. Accordingly, in an independent cohort, patients with a high expression of miR-497 or miR-195 showed higher event-free survival. We identified promoter methylation as the mechanism responsible for miR-497~195 downregulation, as it was associated with low miR-497~195 levels in xenografts and treatment with the demethylating agent Decitabine increased miR-497~195 expression in BCP-ALL cell lines. To study the role of miR-497~195 in BCP-ALL, we stably overexpressed the cluster in three xenograft samples. MiR-497~195 overexpression impaired engraftment of leukemic cells in NOD/SCID mice, as compared to control cells, prolonging recipient survival. Gene expression profiling and ex vivo analysis of miR-497~195 overexpressing xenograft cells showed that inhibition of cell proliferation is a mechanism responsible for the miRNA cluster tumor suppressive role. Mechanistically, CDK4 and CCND3 were downregulated at mRNA and protein levels upon miR-497~195 overexpression, leading to decreased RB1 phosphorylation, reduced transcription of E2F target genes, and inhibition of the entry in S phase. CDK4 mediated G1/S transition is also inhibited by CDKN2A and CDKN2B, which are deleted in 20% of BCP-ALL. Deletion of CDKN2A/B and concomitant low miR-497~195 expression were particularly associated with a high proportion of early relapse cases in our cohort, indicating that impaired regulation of G1/S transition is critical for ALL aggressiveness. Importantly, targeting this pathway with the CDK4/CDK6 inhibitor Palbociclib impaired ex vivo growth of BCP-ALL xenograft samples, indicating a possible therapeutic strategy. Altogether, we showed that the tumor-suppressive cluster miR-497~195 is downregulated by epigenetic repression in BCP-ALL and that low expression is associated with early relapse and rapid engraftment. Low miR-497~195 expression might cooperate with deletion of CDKN2A/B promoting tumor progression, through loss of CCND3/CDK4-dependent control of cell cycle regulation. Citation Format: Elena Boldrin, Enrico Gaffo, Judith Boer, Salih Demir, Julia Zinngrebe, Rainer Claus, Christoph Plass, Monique L. den Boer, Klaus-Michael Debatin, Geertruy te Kronnie, Stefania Bortoluzzi, Lüder Hinrich Meyer. MicroRNA-497~195 cluster suppresses acute lymphoblastic leukemia growth by targeting CCND3/CDK4 and inhibiting cell cycle progression [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2541.

Abstract 531: AT1412, a patient-derived antibody in development for the treatment of CD9-positive precursor B-acute lymphoblastic leukemia

Abstract Introduction Despite rapid advances in immunotherapeutic options for precursor B-acute lymphoblastic leukemia (BCP-ALL), outcomes remain poor especially for adult ALL and relapsed pediatric ALL. With conventional chemotherapy remission percentages in adult ALL range from 75 to 90%, but relapse rates are high and long-term leukemia-free survival ranges between 35-70% depending on age and risk group. The introduction of CD19-targeting immunotherapy has significantly improved patient outcomes in (relapsed) BCP-ALL. However, tumor escape via downregulation of CD19 occurs in a significant number of patients. An ongoing medical need remains for the identification of alternative immunotherapeutic targets for treatment of ALL. Methods CD9 is expressed in 60-80% of BCP-ALL, is correlated with adverse prognosis and has been proposed as therapeutic target for BCP-ALL. AT1412 is a fully human CD9-targeting antibody, that was identified from a patient cured of metastatic melanoma after adoptive T-cell therapy, using our B-cell immortalization technology (AIMSelect) [Kwakkenbos et al. Nat. Med. 2010]. The antibody was selected based on differential binding to melanoma cells as compared to healthy melanocytes and was shown to be successful in killing melanoma cells in vitro and in vivo. Results Binding of AT1412 to BCP-ALL cell lines SUP-B15, MHH-CALL-2 and CCRF-SB varied as expected based on CD9 levels that we detected using a commercial CD9 antibody. AT1412-induced antibody dependent cellular cytotoxicity (ADCC) and antibody dependent cellular phagocytosis (ADCP) correlated with the level of AT1412 binding. No binding was seen to the T-ALL cell line Jurkat. These findings were confirmed in primary ALL samples, obtained prospectively at diagnosis from a cohort of patients with T- or B-ALL (n=38). AT1412 showed binding to the majority of B-ALL samples but not to T-ALL samples. AT1412 induced ADCC of nearly all B-ALL samples it bound to (10/11) and of none of the non-binding B-ALL or T-ALL samples. Cytotoxicity significantly correlated with the level of AT1412 binding. These findings were corroborated by the observation that AT1412 induced specific B-ALL cell death when a bone marrow sample from a newly diagnosed BCP-ALL patient was incubated with AT1412. Remarkably, AT1412 induced cell death in the absence of added effector cells or other (chemo)therapeutic agents, while the sample contained over 80% blasts and as little as 3% lymphocytes. We are currently investigating the in vivo efficacy of the antibody in a humanized immune system mouse model with human BCP-ALL. Conclusion Taken together, the majority of BCP-ALL express CD9 and this is associated with poor prognosis. Our data demonstrate that CD9 can be successfully targeted by the human CD9 antibody AT1412, suggesting that AT1412 has the potential to be developed as a therapeutic antibody for B-ALL. AT1412 is currently being advanced through preclinical development. Citation Format: Greta de Jong, Sophie E. Levie, Remko Schotte, Wouter Pos, Daniel Go, Etsuko Yasuda, Madalina Cercel, Susan E. van Hal, Esmay Frankin, Aniko Szabo, Martijn Kedde, Els Verdegaal, Julien Villaudy, Sjoerd van der Burg, Pauline M. van Helden, Hans van Eenennaam, Hergen Spits, Anita van Rijneveld, Mette D. Hazenberg. AT1412, a patient-derived antibody in development for the treatment of CD9-positive precursor B-acute lymphoblastic leukemia [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 531.

Fast, In Vivo Model for Drug-Response Prediction in Patients with B-Cell Precursor Acute Lymphoblastic Leukemia.

Only half of patients with relapsed B-cell precursor (BCP) acute lymphoblastic leukemia (ALL) currently survive with standard treatment protocols. Predicting individual patient responses to defined drugs prior to application would help therapy stratification and could improve survival. With the purpose to aid personalized targeted treatment approaches, we developed a human–zebrafish xenograft (ALL-ZeFiX) assay to predict drug response in a patient in 5 days. Leukemia blast cells were pericardially engrafted into transiently immunosuppressed Danio rerio embryos, and engrafted embryos treated for the test case, venetoclax, before single-cell dissolution for quantitative whole blast cell analysis. Bone marrow blasts from patients with newly diagnosed or relapsed BCP-ALL were successfully expanded in 60% of transplants in immunosuppressed zebrafish embryos. The response of BCP-ALL cell lines to venetoclax in ALL-ZeFiX assays mirrored responses in 2D cultures. Venetoclax produced varied responses in patient-derived BCP-ALL grafts, including two results mirroring treatment responses in two refractory BCP-ALL patients treated with venetoclax. Here we demonstrate proof-of-concept for our 5-day ALL-ZeFiX assay with primary patient blasts and the test case, venetoclax, which after expanded testing for further targeted drugs could support personalized treatment decisions within the clinical time window for decision-making.

Biomarker Profile for Prediction of Patient Response to Smac Mimetic Monotherapy in Pediatric Precursor B-Cell Acute Lymphoblastic Leukemia

Acute lymphoblastic leukemia (ALL) is the most common malignancy in childhood. While improved multi-agent chemotherapy regimens with individualized risk stratification have led to increased survival rates of approximately 80 percent, 20 percent of patients respond poorly to therapy or relapse. Therefore, novel therapeutic avenues are urgently needed to improve treatment outcome, overcome resistance and reduce side effects. Failure to undergo cell death represents a key survival mechanism of cancer cells and results in drug resistance and clonal escape. Since inhibitor of apoptosis proteins (IAPs) are often overexpressed in malignant cells and their overexpression correlates with inferior survival rates, they provide an attractive molecular target for therapeutic intervention. Small molecule inhibitors have been developed that act as SMAC mimetics (SMs) to counteract the cell death inhibitory function of IAPs. SMs can activate and/or modulate cell death pathways, and are currently being evaluated in clinical trials. Their successful therapeutic implementation requires identification of patients who could benefit from a SM-based treatment regimen ideally before start of therapy. Here, we analyzed the intrinsic activity of two monovalent (AT406 and LCL161) and two bivalent (Birinapant or BV6) SMs on 29 unselected patient-derived pediatric precursor B-cell (BCP)-ALL samples and identified a subset of BCP-ALL primografts to be sensitive to SM treatment (n=8). When we compared gene expression of SM-sensitive (n=8) and SM-insensitive (n=6) patient-derived BCP-ALL samples, we identified a characteristic gene expression signature with 127 differentially regulated genes, amongst them upregulation of TNFRSF1A (TNFR1) in the SM-sensitive subset. In line with previous reports, we confirmed a critical role of the TNF/TNFR1-axis for SM-induced cell death in BCP-ALL by functional analysis. Expression of TNFRSF1A alone, however, did not correlate with sensitivity to SM-induced cell death indicating that TNFR1 is not the only factor regulating cell fate decisions in response to SM treatment. To identify potential biomarker genes for prediction of patient response to SM monotherapy in BCP-ALL, we compared differentially regulated genes of SM responders and non-responders from our cohort with data from a published cohort. Interestingly, we found 4 genes to overlap between these two cohorts. Of these 4 genes TSPAN7, FAM69C, and TNFRSF1A were upregulated whereas MTX2 was downregulated in SM-sensitive samples. The signature identified may reflect a particular TNF network. Analysis of expression levels of these 4 genes in BCP-ALL cell lines (Nalm6, Reh, UoCB6 and RS4;11) revealed that Reh cells, sensitive to SM-induced cell death, exhibited the biomarker profile of primograft sensitivity, i.e. upregulation of TSPAN7, FAM69C, TNFRSF1A and downregulation of MTX2. Nalm6 cells resembled the expression pattern of SM-insensitive samples with a downregulation of TSPAN7, FAM69C, TNFRSF1A and an upregulation of MTX2 and were resistant to SM-induced cell death. RS4;11 and UoCB6 cells showed no pattern. Based on these findings we hypothesized that the respective expression patterns of TSPAN7, FAM69C, TNFRSF1A and MTX2 could predict sensitivity to SMs. An extended screen of additional primary BCP-ALL samples for their expression levels of TSPAN7, FAM69C, TNFRSF1A and MTX2 and response to SMs substantiated this hypothesis. In summary, the subset of primary BCP-ALL samples with sensitivity to SMs is characterized by a gene signature with MTX2 low and TSPAN7, FAM69C and TNFRSF1A high. By using this expression profile, sensitivity to SMs in BCP-ALL could be identified in cell lines and additional primografts. Based on these results, we suggest the identified gene expression pattern as a biomarker for selecting patients to be treated by SM monotherapy in clinical trials. Disclosures No relevant conflicts of interest to declare.

Fibroblast growth factor receptor signaling in pediatric B-cell precursor acute lymphoblastic leukemia

The FGF receptor signaling pathway is recurrently involved in the leukemogenic processes. Oncogenic fusions of FGFR1 with various fusion partners were described in myeloid proliferative neoplasms, and overexpression and mutations of FGFR3 are common in multiple myeloma. In addition, fibroblast growth factors are abundant in the bone marrow, and they were shown to enhance the survival of acute myeloid leukemia cells. Here we investigate the effect of FGFR stimulation on pediatric BCP-ALL cells in vitro, and search for mutations with deep targeted next-generation sequencing of mutational hotspots in FGFR1, FGFR2, and FGFR3. In 481 primary BCP-ALL cases, 28 samples from 19 unique relapsed BCP-ALL cases, and twelve BCP-ALL cell lines we found that mutations are rare (4/481 = 0.8%, 0/28 and 0/12) and do not affect codons which are frequently mutated in other malignancies. However, recombinant ligand FGF2 reduced the response to prednisolone in several BCP-ALL cell lines in vitro. We therefore conclude that FGFR signaling can contribute to prednisolone resistance in BCP-ALL cells, but that activating mutations in this receptor tyrosine kinase family are very rare.

Stearoyl-CoA Desaturase (SCD) Enhances Central Nervous System Leukemia

Background: Central nervous system (CNS) involvement by acute lymphoblastic leukemia (ALL) is a major clinical concern. Leukemic cells can be found in the CNS at diagnosis (1-2%) or, more frequently, at relapse (30%). Very little is known about the pathogenesis and therefore there are no targeted therapies. Prophylactic CNS-directed conventional intrathecal chemotherapy or irradiation are required for relapse-free survival. However, they are associated with substantial rates of short and long term toxicity. Therefore, elucidation of molecular mechanisms and pathways mediating leukemia-cell entry and survival in the CNS is needed to develop alternative CNS-directed treatment strategies. Previous studies showed an increased expression of Stearoyl-CoA desaturase (SCD), a key enzyme of the de novo fatty acid synthesis pathway, in B cell precursor (BCP) ALL cells isolated from cerebrospinal fluid (CSF) of patients at the time of CNS relapse. A small SCD positive population was detected in the bone marrow (BM) at leukemia diagnosis in patients who later developed isolated CNS relapse, defining a potential biomarker for CNS relapse. It is unknown, however, if SCD has a functional role in CNS leukemia.Aim: To examine the hypothesis that increased expression of SCD enhances trafficking and survival of human B-ALL cells in the CNSMethods: We analyzed leukemia-cell entry into the CNS using xenografts of human BCP-ALL cell lines. Microarray profile of cells isolated from CNS and BM of transplanted mice was performed. Cell lines were transduced to overexpress human SCD and evaluated in vitro for proliferation kinetics and metabolic SCD activity. In vivo, SCD overexpressing cells were transplanted in NSG mice,sacrificed upon the first symptoms of CNS involvement, e.g. hind limb paralysis. BM, spleen and meninges were collected and analyzed to check human engraftment by FACS. The tumor load was expressed as total amount of leukemic cells in each organ. Competition assays were performed by transplanting SCD overexpressing and WT cells in the same mouse in a 1:1 ratio.Results: BCP-ALL cells transplanted into NSG mice faithfully recapitulated pathological features of meningeal infiltration seen in patients with ALL. Gene expression analysis of cells collected from BM and meninges of leukemic mice revealed up-regulation of the genes belonging to the signaling pathway of sterol regulatory element binding proteins (SREBPs) in ALL cells isolated from the CNS. SCD, whose transcription is controlled by the SREBP family, was significantly upregulated. SCD overexpression did not alter proliferation in vitro. Since SCD introduces a double bond in Stearoyl-CoA, its activity was measured as the ratio of unsaturated/saturated fatty acids in the cells. That ratio was increased in SCD overexpressing cells in vitro, confirming the functionality of the enzyme. In vivo, mice transplanted with SCD overexpressing cells led to a faster onset of CNS disease manifested by a clinical phenotype of earlier hind limb paralysis compared to control and significant increased number of leukemic cells in the CNS (Figure 1A).SCD overexpression also induced CNS engraftment of another B-ALL cell line, REH, which is not naturally prone to invade the central nervous system. Mice transplanted with SCD overexpressing REH cells showed the same phenotype of earlier hind limb paralysis and accumulation of leukemic cells in the CNS as the CNS-prone 018z cells, while WT REH did not show any CNS engraftment but comparable tumor load in BM and spleen (Figure1B). To reproduce the clonal heterogeneity in SCD expression observed previously in patients' BM, we performed a competition assay transplanting SCD overexpressing cells and control cells, expressing different fluorochromes, in the same mouse in a 1:1 ratio. In the CNS, the ratio between SCD overexpressing and WT cells ranged from 2 to 20 fold. This effect was unique to the CNS and not reproducible in the other hematopoietic organs where the 1:1 ratio was maintained (Figure 1C). Moreover, SCD overexpression sensitized leukemic cells to mTOR inhibitors, suggesting a potential therapeutic optionConclusion: SCD has a role in homing and survival of leukemic cells in the CNS and may be used as early predictor of CNS relapse. This study reveals a role for SCD and fatty acid metabolism in the pathogenesis of CNS leukemia suggesting that this pathway maybe targeted for specific therapy of this devastating disease. [Display omitted] DisclosuresHalsey:Jazz Pharmaceuticals: Honoraria, Other: Support for conference attendance.

Lack of association between deletion polymorphism of BIM gene and in vitro drug sensitivity in B-cell precursor acute lymphoblastic leukemia

A deletion polymorphism in the BIM gene was identified as an intrinsic mechanism for resistance to tyrosine kinase inhibitor in chronic myeloid leukemia patients in East Asia. BIM is also involved in the responses to glucocorticoid and chemotherapy in acute lymphoblastic leukemia (ALL), suggesting a possible association between deletion polymorphism of BIM and the chemosensitivity of ALL. Thus, we analyzed 72 B-cell precursor (BCP)-ALL cell lines established from Japanese patients. Indeed, higher BIM gene expression was associated with good in vitro sensitivities to glucocorticoid and chemotherapeutic agents used in induction therapy. We also analyzed the methylation status of the BIM gene promoter by next generation sequencing of genome bisulfite PCR products, since genetic polymorphism could be insignificant when epigenetically inactivated. Hypermethylation of the BIM gene promoter was associated with lower BIM gene expression and poorer sensitivity to vincristine. Of note, however, the prevalence of a deletion polymorphism was not associated with the BIM gene expression level or drug sensitivities in BCP-ALL cell lines, in which the BIM gene was unmethylated. These observations suggest that an association of a deletion polymorphism of BIM and the response to induction therapy in BCP-ALL may be clinically minimal.

FOXO1 Is Involved in the Regulation of B-Cell Precursor Acute Lymphoblastic Leukemia Survival and Serves As a Novel Target for Directed Therapy

Acute lymphoblastic leukemia (ALL) is the most common pediatric and adolescent malignancy. Although current treatment provides five-year event-free survival, in up to 20% conventional chemotherapy fails resulting in relapse with inferior prognosis.FOXO1 is a member of the forkhead family of transcription factors, which is preferably expressed in B-cells with high expression at the early B-cell stage. FOXOs are involved in several cellular processes including cell death and proliferation, anti-cancer drug resistance and protection from oxidative stress. Since FOXO1 can enhance tumor growth and potentiate metastasis, we aimed to investigate the effects of FOXO1 inactivation on B-cell precursor (BCP)-ALL, including preclinical in vivo evaluation.FOXO1 expression levels were compared among 497 cancer samples using the Genevestigator online software. Expression of FOXO1 in BCP-ALL was significantly higher than in any of the other cancer types. Next, we investigated FOXO1 expression and subcellular localization in 3 BCP-ALL cell lines by cellular fractionation and fluorescent microscopy. Both methods showed localization of FOXO1 in the nucleus, indicating transcriptionally active FOXO1 in BCP-ALL.In order to study the potential anti-tumor effect of FOXO1 repression, we investigated genetically modified, FOXO1 deficient BCP-ALL cell lines (n=5) and observed no cell death induction in control transduced cells, in contrast to a clear reduction of cell viability of up to 80% upon FOXO1 knock-down, clearly indicating dependency of BCP-ALL cells on FOXO1. Moreover, lentiviral mediated FOXO1 knockdown did not induce cell death in the Hodgkin's lymphoma cell line cHL, suggesting a BCP-ALL specific importance for FOXO1.Based on these results indicating the importance of FOXO1 expression for BCP-ALL maintenance, we investigated the feasibility of pharmacological interference with FOXO1. Exposure of 7 BCP-ALL, 4 T-ALL, 3 B-cell NHL, 2 DLBCL and 3 cHL cell lines to the small molecular weight FOXO1 inhibitor AS1842856 showed effectivity in BCP-ALL lines, reflected by significantly higher half maximal inhibitory concentrations (IC50) by MTT test. The most sensitive cell line was the BCP-ALL line RS4;11, while the cHL cell line SUP-HD1 showed insensitivity for FOXO1 inhibition (IC50: 3 nM and 26 µM), again indicating that BCP-ALL is particularly dependent on FOXO1 activity. Caspase 3 cleavage detected upon exposure to AS1842856 showed induction of apoptosis as mechanism of cell death.Furthermore, we evaluated the sensitivity of primary BCP-ALL primograft samples (n=9) exposing the ALL cells to increasing pharmacologically relevant concentrations of AS1842856. The inhibitor increased cell death as measured by flow cytometry (FSC/SSC criteria) in all of the samples tested in a time and dose dependent manner. Importantly, FOXO1 inhibition also showed activity on high risk leukemias including MLL-rearranged and early or second-relapse cases.Moreover, we investigated the in vivo effectivity of AS1842856. Two different patient derived leukemias were transplanted onto NOD/SCID mice and upon leukemia manifestation vehicle or AS1842856 was administered for a time of 11 days. At the end of the experiment, all mice were sacrificed and tumor loads were quantified in spleen, bone marrow and central nervous system (CNS). Importantly, tumor loads of all compartments and spleen sizes were significantly reduced in AS1842856 treated animals (p=0.028, U-test). Moreover, in an early-relapse sample leukemia-free survival upon AS1842856 treatment was evaluated. Mice were treated by vehicle or AS1842856 (n=10/group) during 11 days. Leukemia-free survival was significantly prolonged in mice which received AS1842856 (p=0.003, Log-rank test).Taken together, we show that the active form of FOXO1 is highly expressed in BCP-ALL cells as compared to other cancers, and that viability of BCP-ALL cells is regulated by FOXO1 activity. Importantly, silencing or pharmacological inhibition of FOXO1 induces cell death in BCP-ALL primogafts including high risk cases, both ex vivo and preclinically in vivo. Thus, targeting FOXO1 provides a promising novel strategy for therapeutic intervention in these high-risk subtypes of BCP-ALL. DisclosuresNo relevant conflicts of interest to declare.

Lysosomal Cell Death and Apoptosis Crosstalk - Synergistic Role in Bcl-2 Inhibitor (ABT-263) Mediated Cell Death in B-Cell Precursor Acute Lymphoblastic Leukemia

Although improved treatment of pediatric Acute Lymphoblastic Leukemia (ALL) has become increasingly successful with cure rates of up to 90%, leukemia reoccurrence is clearly associated with inferior prognosis. Treatment failure and resistance in leukemias are considered to involve defects in apoptosis signaling, which forms the basis for apoptogenic treatment approaches such as the use of Bcl-2 inhibitors. Identification of alternative cell death programs like lysosomal membrane permeabilization-induced cell death may provide a novel strategy for the treatment of ALL. Previous studies showed that lysosomal activity, as well as their traffic properties are greatly altered during tumorigenesis. Intralysosomal hydrolases such as cathepsin B, L and D have been implicated in cancer progression and high expression levels are generally associated with poor prognosis. Lysosomotropic agents modulating lysosomal integrity may overcome cell death resistance and might therefore also improve the outcome of high risk or ALL relapse patients.In this study, we investigated the efficacy of the lysosomotropic compound B10, a derivative of Betulinic acid, as well as its combination with the Bcl-2 inhibitor ABT-263 in B-cell precursor- (BCP-) ALL.In BCP-ALL cell lines, B10 showed induction of cell death combined with increased DNA fragmentation. Interestingly, additional treatment with the pan-caspase inhibitor zVAD.fmk only partially rescued B10 triggered loss of cell viability, indicating that B10 is not exclusively inducing caspase-dependent apoptosis, but also induces an additional alternative cell death program. B10 permeabilized lysosomes as indicated by the significant decrease of LysotrackerRED positive populations detected by flow cytometry, and treatment with E-64d, a potent inhibitor of thiol proteases and cathepsins, reduced B10-induced cell death, thus emphasizing the cathepsin-dependent effect of B10. To exclude that B10-induced lysosomal permeabilization might represent a secondary effect, a panel of different substances was tested for their activity on lysosomal membrane integrity. While treatment with dexamethasone, chloroquine, vincristine and asparaginase also showed lysosomal permeabilization, co-treatment with E-64d did not reduce cell death indicating a secondary effect of these compounds on lysosomes. In addition to cell lines, we tested the effect of B10 on 15 primary leukemia samples isolated from ALL bearing mice of established patient-derived NOD/SCID/hu BCP-ALL xenografts. In a majority of these individual patient-derived leukemias, B10 induced cell death that could be inhibited by E-64d, thus demonstrating a lysosomal protease-dependent death program also in primary ALL.In addition to its classical role in triggering the intrinsic mitochondria dependent apoptosis pathway, the pro-apoptotic Bcl-2 family member Bax was previously described to be involved in the regulation of lysosomal membrane integrity, pointing to a possible synergistic effect of B10 and BH3-mimetics. Intriguingly, in cell fractionation assays we observed increased Bax recruitment to lysosomal membranes when B10 is present. Interestingly, co-treatment with the Bcl-2 inhibitor ABT-263, which displaces Bcl-2 from its inhibitory binding to Bax, led to increased lysosomal permeabilization, loss of mitochondrial membrane potential, and caspase activation indicating involvement of the intrinsic apoptosis pathway. The importance of lysosomal Bax recruitment for the activity of ABT-263 and its concomitant effect on lysosomal membrane permeabilization was further supported by Bax knockdown experiments, since induction of lysosomal disruption, release of cathepsins and their subsequent effect on cell death activation by B10 was reduced.Taken together, our findings suggest an important role of lysosomal membrane permeabilization-induced cell death for the activity of Bcl-2 inhibitors such as ABT-263. The combination of BH3 mimetics with lysosomotropic compounds may provide the basis for novel molecular directed treatment strategies. DisclosuresNo relevant conflicts of interest to declare.

Involvement of Allele-Specific Methylation of Asparagine Synthetase Gene in Asparaginase Sensitivity of BCP-ALL

Asparaginase is one of the most important components for the treatment of ALL. ALL cells are supposed to be unable to synthesize adequate amounts of Asparagine (Asn), and, therefore, depend on extracellular source of Asn to survive. Asparaginase therapy induces the depletion of serum Asn by catalyzing the deamination of Asn and leads to cell death of ALL cells. Asparagine synthetase (ASNS) is an enzyme that produces Asn from Aspartic acid. Thus, silencing of the ASNS gene in ALL cells could be crucial for complete starving ALL cells of the Asn. Considering that the ASNS gene has a CpG island in its promotor, aberrant methylation of CpG island could be one of epigenetic mechanisms for silencing of ASNS gene in ALL cells. Previous qualitative analysis of ALL samples using methylation-specific restriction enzyme revealed frequent methylation of CpG island in the ASNS gene. However, associations of methylation status of ASNS gene with its expression level and sensitivity to asparaginase in ALL cells remain unknown. Moreover, little is known about mechanisms for leukemia-specific ASNS gene silencing by methylation. To shed light on these issues, we analyzed a large panel of BCP-ALL cell lines. We quantified ASNS gene expression level by real time RT-PCR in 79 BCP-ALL cell lines cultured in the presence or the absence of L-asparaginase (L-asp), and determined IC50 values of L-asp using alamar blue assay. In the majority of cell lines, although degree of the induction was highly variable, ASNS gene expression level was upregulated in the presence of L-asp. IC50 value of L-asp showed significant correlation with ASNS gene expression level cultured in the presence of L-asp (r=0.222, p=0.049) rather than that in the absence of L-asp (r=0.193, p=0.089). We next analyzed methylation status of the ASNS gene in 79 BCP-ALL cell lines by bisulfite PCR sequencing using a next-generation sequencer (NGS). Strong correlation was confirmed between mean % methylation by NGS and Sanger sequencing in representative cell lines. Of importance, mean % methylation in 79 BCP-ALL cell lines showed significant negative correlation with ASNS gene expression level cultured in the presence of L-asp (r=-0.482, p=6.73x10-6) and, subsequently, IC50 value of L-asp (r=-0.39, p=3.86x10-4). Unexpectedly, % methylation of 79 cell lines distributed in three clusters; 15 cell lines (19%) were highly methylated (>66%, median; 89%), 26 cell lines (32.9%) were moderately methylated (33-66%, median; 40%), and 38 cell lines (48.1%) were weakly methylated (<33%, median; 3.7%). In the majority of moderately methylated cell lines, histograms of % methylation in each read of NGS showed two peaks of high and low methylation, suggesting an allele-specific methylation. In the middle of CpG island, tandem repeat polymorphism of 14bp nucleotides is located adjacent to methylation-specific restriction enzyme site of Aor13HI. Of note, in 7 out of 8 moderately methylated cell lines with heterozygous tandem repeat genotype, only single PCR product was detectable when PCR was performed after Aor13HI treatment, whereas two PCR products derived from two- and three-repeat alleles was detectable when PCR was performed without treatment, indicating an allele-specific methylation. We next analyzed a possible one-allele-loss of the ASNS gene in highly methylated (>66%; 8 cell lines) and weakly methylated (<20%; 12 cell lines) cell lines. We directly sequenced genotype in a portion of introns 2 and 4 and exon 5 based on the imputated SNP genotypes, and confirmed heterozygous genotype in every cell lines at least in one of eight SNPs analyzed, demonstrating that loss-of-heterozygosity is not the mechanism for high or low methylation of the ASNS gene. Similar pattern of methylation was observed in 52 BCP-ALL samples. Taken together, these observations indicate that stepwise allele-specific methylation of ASNSgene is critically involved in the sensitivity to L-asp of BCP-ALL. DisclosuresNo relevant conflicts of interest to declare.

Unique long non-coding RNA expression signature in ETV6/RUNX1-driven B-cell precursor acute lymphoblastic leukemia.

Overwhelming evidence indicates that long non-coding RNAs have essential roles in tumorigenesis. Nevertheless, their role in the molecular pathogenesis of pediatric B-cell precursor acute lymphoblastic leukemia has not been extensively explored. Here, we conducted a comprehensive analysis of the long non-coding RNA transcriptome in ETV6/RUNX1-positive BCP-ALL, one of the most frequent subtypes of pediatric leukemia. First, we used primary leukemia patient samples to identify an ETV6/RUNX1 specific expression signature consisting of 596 lncRNA transcripts. Next, integration of this lncRNA signature with RNA sequencing of BCP-ALL cell lines and lncRNA profiling of an in vitro model system of ETV6/RUNX1 knockdown, revealed that lnc-NKX2-3-1, lnc-TIMM21-5, lnc-ASTN1-1 and lnc-RTN4R-1 are truly regulated by the oncogenic fusion protein. Moreover, sustained inactivation of lnc-RTN4R-1 and lnc-NKX2-3-1 in ETV6/RUNX1 positive cells caused profound changes in gene expression. All together, our study defined a unique lncRNA expression signature associated with ETV6/RUNX1-positive BCP-ALL and identified lnc-RTN4R-1 and lnc-NKX2-3-1 as lncRNAs that might be functionally implicated in the biology of this prevalent subtype of human leukemia.

Synergistic Activity of ABT-199 with Conventional Chemotherapy and Dinaciclib in B-Cell Precursor Acute Lymphoblastic Leukemia

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. DisclosuresNo relevant conflicts of interest to declare.

Targeting Mutant p53 in Pediatric Acute Lymphoblastic Leukemia

Mutations of the tumor suppressor gene TP53 have been described to be associated with aggressive disease and inferior prognosis in different types of cancer, including hematological malignancies. In acute lymphoblastic leukemia (ALL), TP53 alterations are infrequently found at diagnosis but have recently been described in about 12% of patients at relapse. This suggests an association with therapy resistance in high risk/relapsed ALL and patients with TP53 mutated ALL have in fact an inferior outcome. Small molecule compounds targeting mutated TP53 such as APR-246, initially described as PRIMA-1MET (p53-dependent reactivation and induction of massive apoptosis) leading to apoptosis induction have shown activity in several types of malignancies with mutated TP53. In ALL, however, mutant TP53 has so far not been addressed as a target for therapeutic intervention.In this study, we investigated a large cohort of patient-derived pediatric B cell precursor (BCP)-ALL primograft samples to identify cases with mutated TP53. Further, we analyzed the effects of APR-246 and evaluated its activity on BCP-ALL cell lines and primografts with mutated (mut) orwild type (wt) TP53.Altogether, 62 BCP-ALL primograft samples established from patients at diagnosis (n=53) or relapse (n=9) by transplantation of primary ALL cells onto NOD/SCID mice were screened for TP53 mutations by denaturating high-performance liquid chromatography (dHPLC) followed by Sanger sequencing of exons 4 to 10 to confirm detected mutations. We identified 4 cases with TP53 mut, 3 obtained from diagnosis (5.6%) and one at relapse (11.1%), corresponding to frequencies described in clinical studies. Mutated cases were further analyzed by fluorescence in situ hybridization (FISH), revealing a 17p deletion in one TP53 mut sample. Similarly, we analyzed 6 BCP-ALL cell lines and identified 2 TP53 mut and 4 TP53 wt lines.Exposure of BCP-ALL primograft (TP53 mut n=4, TP53 wt n=4) and cell line (TP53 mut n=2, TP53 wt n=4) samples to the DNA damaging agent doxorubicin showed, as expected, resistance of TP53 mut leukemia cells for cell death induction, reflected by significantly higher half maximal inhibitory concentrations (IC50; TP53 mut 49 and 143 ng/ml, TP53 wt mean 12 ng/ml) and lower induction of cell death (TP53 mut 16 to 23%, TP53 wt 10 to 60%) in TP53 mut ALL, corroborating the tumor-suppressive function of p53 in ALL.We then investigated the sensitivity of BCP-ALL cell lines for cell death induction by APR-246 (kindly provided by Aprea, Stockholm, Sweden). We observed high sensitivity for APR-246 in TP53 mut (IC50: 5 µM for both cell lines) as compared to TP53 wt ALL (mean IC50: 58 µM). DNA fragmentation and Annexin-V/propidium-iodide (PI) positivity revealed apoptosis as mechanism of APR-246 mediated cell death. Reactive oxygen species (ROS) have recently been described to mediate APR-246 induced cell death in multiple myeloma cells. Therefore, we investigated ROS levels by detection of oxidation-specific fluorescence of dichlorodihydrofluorescein diacetate (DCFDA) in ALL cells. Interestingly, ROS quenching by N-acetyl cysteine abolished induction of cell death in TP53 mut but not TP53 wt ALL cells indicating ROS as a mediator of APR-246 induced cell death in TP53 mut ALL. Furthermore, we addressed p53 activation in response to APR-246 by assessing phosphorylation of p53 (p53pSer15) using phosphoflow cytometry. Most interestingly, APR-246 led to 6-fold increased p53pSer15 levels in TP53 mut compared to no activation in TP53 wt leukemia cells, indicating restoration of p53function upon APR-246treatment in BCP-ALL.Based on these findings, we addressed the effectivity of APR-246on primary, patient-derived primografts and compared sensitivities for cell death induction in TP53 mut (n=4) and TP53 wt (n=4) samples. Importantly, the pattern of responsiveness of TP53 mut ALL was also identified in TP53 mut patient-derived ALL samples with induction of significantly higher cell death rates in TP53 mut ALL (TP53 mut 48%, TP53 wt 18%, 5 µM APR-246, 24 h).Taken together, we showed that TP53 mut BCP-ALL can be targeted by APR-246 leading to re-activation of p53, induction of ROS dependent apoptosis and effective leukemia cell killing. Thus, targeting and re-activation of mutated p53 provides a promising novel strategy for therapeutic intervention in this high-risk subtype of BCP-ALL. DisclosuresSelivanova:Aprea: Patents & Royalties: APR-246. Tausch:Gilead: Other: Travel support. Stilgenbauer:Gilead: Honoraria, Research Funding.