pre-B ALL Cell Lines Research Articles

1,5-Anhydroglucitol promotes pre-B acute lymphocytic leukemia progression by driving glycolysis and reactive oxygen species formation

BackgroundPrecursor B-cell acute lymphoblastic leukemia (pre-B ALL) is the most common hematological malignancy in children. Cellular metabolic reorganization is closely related to the progression and treatment of leukemia. We found that the level of 1,5-anhydroglucitol (1,5-AG), which is structurally similar to glucose, was elevated in children with pre-B ALL. However, the effect of 1,5-AG on pre-B ALL was unclear. Here, we aimed to reveal the roles and mechanisms of 1,5-AG in pre-B ALL progression.MethodsThe peripheral blood plasma level of children with initial diagnosis of pre-B ALL and that of healthy children was measured using untargeted metabolomic analysis. Cell Counting Kit-8 assay, RNA sequencing, siRNA transfection, real-time quantitative PCR, and western blot were performed using pre-B ALL cell lines Reh and HAL-01. Cell cycle, cell apoptosis, ROS levels, and the positivity rate of CD19 were assessed using flow cytometry. Oxygen consumption rates and extracellular acidification rate were measured using XFe24 Extracellular Flux Analyzer. The lactate and nicotinamide adenine dinucleotide phosphate levels were measured using kits. The effect of 1,5-AG on pre-B ALL progression was verified using the In Vivo Imaging System in a xenotransplantation leukemia model.ResultsWe confirmed that 1,5-AG promoted the proliferation, viability, and intracellular glycolysis of pre-B ALL cells. Mechanistically, 1,5-AG promotes glycolysis while inhibiting mitochondrial respiration by upregulating pyruvate dehydrogenase kinase 4 (PDK4). Furthermore, high levels of intracellular glycolysis promote pre-B ALL progression by activating the reactive oxygen species (ROS)-dependent mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway. Conversely, N-acetylcysteine or vitamin C, an antioxidant, effectively inhibited 1,5-AG-mediated progression of leukemia cells.ConclusionsOur study reveals a previously undiscovered role of 1,5-AG in pre-B ALL, which contributes to an in-depth understanding of anaerobic glycolysis in the progression of pre-B ALL and provides new targets for the clinical treatment of pre-B ALL.

Synergistic apoptotic effect of Mcl-1 inhibition and doxorubicin on B-cell precursor acute lymphoblastic leukemia cells.

Myeloid cell leukemia-1 (MCL-1) is a component of the Bcl-2 anti-apoptotic family that plays a key role in cell proliferation and differentiation. Despite tremendous improvements toward identification of the role of MCL-1 in leukemia progression, the functional significance and molecular mechanism behind the effect of MCL-1 overexpression on the proliferation of B-cell precursor acute lymphoblastic leukemia (BCP-ALL) has not been clarified. In addition, less well appreciated is the effect of MCL-1 inhibition on the potentiation of doxorubicin-induced apoptosis in BCP-ALL cell lines. In the present study, we aimed to shed light on the anti-cancer properties of S63845, a potent Mcl-1 inhibitor, in BCP-ALL cell lines either alone or in combination with a chemotherapeutic drug. METHODS ANDRESULTS: Mononuclear cells from patients with Pre-B ALL and BCP-ALL cell lines were treated with S63845 in presence or absence of doxorubicin, induction of apoptosis was evaluated using Annexin-V/PI staining kit. mRNA and protein expression levels were assessed by qRT-PCR and western blot analysis, respectively. Our results declared that inhibition of Mcl-1 impairs cell growth and induces apoptosis in pre-B ALL cells through activation of caspase-3 and up-regulation of a repertoire of pro-apoptotic Bcl-2 family. Additionally, S63845 acts synergically with doxorubicin to induce apoptosis in BCP-ALL cell lines. Our data declared that MCL-1 inhibition alone or in combination with a chemotherapeutic agent is considered an appealing strategy for the induction of apoptosis in BCP-ALL cells.

Optimization of therapeutic T cell expansion in G-Rex device and applicability to large-scale production for clinical use

Our center performs experimental clinical studies with advanced therapy medicinal products (ATMPs) based on polyclonal T cells, all of which are currently expanded in standard T-flasks. Given the need to increase the efficiency and safety of large-scale T cell expansion for clinical use, we have optimized the method to expand in G-Rex devices both cytokine-induced killer cells (CIKs) from peripheral or cord blood and blinatumomab-expanded T cells (BETs). We show that the G-Rex reproducibly allowed the expansion of >30 × 106 CD3+ cells/cm2 of gas-permeable membrane in a mean of 10 to 11 days in a single unit, without manipulation, except for addition of cytokines and sampling of supernatant for lactate measurement every 3 to 4 days. In contrast, 21 to 24 days, twice-weekly cell resuspension and dilution into 48 to 72 T-flasks were required to complete expansions using the standard method. We show that the CIKs produced in G-Rex (CIK-G) were phenotypically very similar, for a large panel of markers, to those expanded in T-flasks, although CIK-G products had lower expression of CD56 and higher expression of CD27 and CD28. Functionally, CIK-Gs were strongly cytotoxic in vitro against the NK cell target K562 and the REH pre-B ALL cell line in the presence of blinatumomab. CIK-Gs also showed therapeutic activity in vivo in the Ph+ pre-B ALL-2 model in mice. The expansion of both CIKs and BETs in G-Rex was validated in good manufacturing practices (GMP) conditions, and we plan to use G-Rex for T cell expansion in future clinical studies.

Inhibition of WHSC1 As a Promising Therapeutic Strategy in Childhood ALL

Childhood acute lymphoblastic leukemia (cALL) is the most frequent pediatric cancer, accounting for ~25% of all pediatric tumors. Nearly 20% of patients do not respond to current treatments, making cALL one of the leading cause of disease-related mortality amongst children. Moreover, sizable portion of childhood leukemia survivors develop significant long-term or late effects of treatment. This rate of refractory/relapse cases and the increased risk of chronic morbidity and early mortality among survivors highlight the pressing need for new treatment strategies in cALL. These can be developed through in-depth investigation of the genetic architecture underlying cALL. For this purpose, we performed whole exome sequencing (WES) of matched tumor-normal pair genomic DNA from 200 cALL patients to identify putative somatic driving mutations. A repertoire of 132 candidate drivers were further validated by performing a targeted knockdown functional screen in 3 pre-B ALL cell lines (NALM6, REH and 697). Through this screen, we identified WHSC1 as putative driver gene. WHSC1 is a methyltransferase that dimethylates lysine 36 on histone H3 (H3K36me2). Eight of the 200 (4%) cALL patients harboured a mutation in WHSC1 (7 pre-B and 1 pre-T ALL). In all 8 cases, the mutation led to a change at amino acid 1099 from a glutamic acid to lysine (E1099K). It has been shown that this activating mutation is associated with global increase in H3K36me2, which is accompanied by substantial changes in gene expression. Transcriptome analysis, using RNA-seq technique, of 6 cALL patients (2 with the mutation and 4 without) revealed distinctive differential expression pattern. We observed a specific enrichment in Wnt pathway genes in presence of the WHSC1 E1099K mutation (6.52 fold, P= 0.05), suggesting a stem cell signature. MCTP39 has been identified as an inhibitor of WHSC1, but has never been tested in ALL. Thus, we treated 4 pre-B ALL cell lines, 3 wild-type for WHSC1 (697, REH and Nalm6) and 1 carrying the mutation E1099K (RS411), as well as the non-leukemic human B cell precursor GM12878 cell line with variable concentrations of MCTP39 and calculated IC50. The IC50 in the leukemic cell lines 697, REH, Nalm6 and RS411 were 2.5µM, 1.7µM, 0.88µM and 0.89µM respectively, whereas it reached 25µM in the non-leukemic GM12878, suggesting a specificity for transformed cells. Although Nalm6 and RS411 had almost the same IC50 at 48h, cell growth curves showed differences in late effect (beyond 3 days), with RS411 being more sensitive to MCTP39. The latter differential effect has been confirmed by colony assay with reduction in plating efficiency of 69% and 45% in RS411 and Nalm6, respectively, when treated 48h with MCTP39 at 0.25µM. This result suggests that MCTP39 targets specifically stem/progenitor cells, particularly those that harbor the mutation WHSC1 E1099K. To confirm that the cellular effect of MCTP39 is due to WHSC1 inhibition, we assessed the impact of MCTP39 on H3K36me2 expression, using fluorometric assay and western blot. In both Nalm6 and RS411cell lines, MCTP39 led to significant decrease in H3K36me2. Then, we analyzed whether MCTP39 synergizes with doxorubicin, an anthracycline that is commonly used in cALL treatment. In both Nalm6 and RS411 cell lines, MCTP39 showed a clear synergistic effect at all concentration tested, permitting 40% reduction in doxorubicin concentration to obtain equal effect. Altogether, this study indicates that inhibition of WHSC1 is a promising treatment strategy in cALL patients, particularly in those carrying WHSC1 mutation. Further investigations are needed to recapitulate these results in vivo and to elucidate the pathways affected as result of the inhibition of H3K36me2 by MCTP39. DisclosuresNo relevant conflicts of interest to declare.

Reciprocal Regulation of BCL6 and MLL Expression As a Vulnerability in MLL -Rearranged Acute Lymphoblastic Leukemia

Background: Chromosomal translocations involving the Mixed Lineage Leukemia gene MLL account for ~70% of infant acute lymphoblastic leukemias (ALLs; Ayton and Cleary, 2001) and are associated with dismal prognosis. These findings underscore the importance of understanding the molecular mechanisms underlying disease progression of MLL-rearranged (MLLr) leukemia for the development of novel therapeutics. Recently, an integrative epigenomic analysis has identified the transcriptional repressor and proto-oncogene BCL6 as a transcriptional target in MLL-AF4 -driven pre-B ALL (Geng et al., 2012). BCL6 functions as a mediator of drug resistance to tyrosine kinase inhibitors in Ph+ ALL (Duy et al., 2011). Here, we tested the hypothesis that BCL6 is required for MLLr leukemia and represents a therapeutic target in MLLr pre-B ALL.Results: Western blot analysis of a panel of patient-derived pre-B ALL cells (MLLr : n= 7; ETV6-RUNX1 : n= 2; BCR-ABL1 : n= 1; not otherwise specified: n= 4) and normal CD19+ B cells (n= 3) revealed that BCL6 levels were elevated in MLL r ALL samples. Furthermore, immunohistochemistry staining in bone marrow biopsies from ALL patients (n= 72) showed that BCL6 levels were upregulated in MLLr cases. Expression of MLL-ENL or MLL-AF4 fusion protein increased Bcl6 protein levels (~10-fold) in murine pre-B cells. Moreover, ChIP-seq analysis of human MLLr pre-B ALL cell lines using antibodies against MLL, AF4 and ENL demonstrated direct binding to the BCL6 promoter. Studying methylation status revealed that the BCL6 promoter was hypomethylated in patients with MLLr pre-B ALL compared to patients with other subtypes of pre-B ALL (including BCR-ABL1, ETV6-RUNX 1 and hyperdiploid) as well as normal pre-B cells from healthy donors (St. Jude, n= 132, Figueroa, 2014). Consistent with these observations, multivariate analysis of high risk pre-B ALL patients showed that high BCL6 levels in combination with MLL rearrangements were associated with the worst clinical outcome (COG P9906). Collectively, our findings demonstrated that BCL6 levels are elevated in MLLr ALL, and that expression levels of MLL and BCL6 can serve as predictors for risk stratification in ALL.Interestingly, expression of wild-type MLL was positively correlated with that of BCL6 in both pediatric (COG P9906) and adult (ECOG E2993) pre-B ALL. Furthermore, multivariate analysis comparing MLLHighBCL6High patients with MLLLowBCL6Low patients showed that high expression levels of both genes correlated with poor clinical outcome (COG P9906). These findings prompted us to study whether MLL regulates BCL6 expression, and vice versa . Cre-mediated deletion of Mll abrogated imatinib-mediated induction of Bcl6 in BCR-ABL1 -driven pre-B ALL, indicating that Mll is required for upregulation of Bcl6 expression. While genetic inactivation of Bcl6 as well as treatment with the BCL6-specific retro-inverso peptide-inhibitor (RI-BPI) decreased Mll expression, doxycycline-induced expression of Bcl6 upregulated Mll expression. Combining chromatin immunoprecipitation and DNA microarray (ChIP-on-chip), we found that BCL6 was recruited to the MLL promoter in pre-B ALL. Taken together, these findings support our proposed scenario of reciprocal regulation of MLL and BCL6 expression.Notably, ChIP-seq analyses of MLLr pre-B ALL cells showed that there are 268 common gene targets of both MLL and BCL6. Among the common targets include cell cycle checkpoint regulators (CDKN1B, ETS2, BMI1, RB1), apoptosis facilitator BIM, and tumor suppressor TET2, indicating that BCL6 is a key regulator of the transcriptional program in MLLr pre-B ALL. In support of an essential role of BCL6 in MLLr ALL, expression of a dominant negative BCL6 mutant in patient-derived MLLr pre-B ALL cells induced rapid depletion from cell culture in competitive-growth assays. Furthermore, RI-BPI treatment resulted in cell cycle arrest, impaired colony forming ability and inhibited leukemia initiation in transplant recipient mice. Importantly, RI-BPI in combination with standard chemotherapy Vincristine exerted more potent effects on killing MLLr pre-B ALL cells than either agent alone.Conclusions: Our findings suggest that aberrant BCL6 expression levels are important for disease progression, and that pharmacological inhibition of BCL6 augments responsiveness to standard chemotherapy in the treatment of MLLr pre-B ALL. DisclosuresHurtz:Incyte: Research Funding. Armstrong:Cyteir: Consultancy; Imago: Consultancy; C4 Therapeutics: Consultancy; Epizyme: Consultancy.

Enhancing the Antigen-Sensitivity of the CD22 CAR through Modulation of the Affinity and Linker-Length of the Single-Chain Fragment Variable

The advent of chimeric antigen receptor (CAR) T cell therapy has revolutionized the treatment of relapsed/refractory acute lymphoblastic leukemia (r/r ALL). CD19 directed CAR T cells have demonstrated the ability to induce complete remissions in up to 90% of r/r ALL patients. Despite this remarkable upfront success, relapse after CAR T cell therapy remains a major obstacle to long term remissions. A major mechanism for relapse after CD19-directed CAR T cell therapy is the recurrence of antigen-negative ALL cells. In recent years, CD22 CAR T cell therapy has emerged as an effective salvage therapy for patients with CD19-negative ALL. In a phase I clinical trial, CD22 CAR T cells were able to induce remission in up to 80% of patients with CD19-negative ALL. Patients achieving remission, who did not undergo a consolidative hematopoietic stem cell transplant, were found to be at high risk of relapse due to downregulation of the CD22 antigen below the threshold required for effective CD22 CAR T cell activity. Thus, strategies to increase the antigen-sensitivity of CD22 CAR T cells have the potential to enhance the induction and duration of remission in ALL patients. As the properties of a CAR that influence sensitivity to antigen are not well defined, we began by testing the impact of increasing the affinity of the single-chain fragment variable (scFv) for the CD22 antigen. T cells from healthy donors were activated and transduced with a second-generation, 4-1BB CAR containing either the standard affinity (SA)-m971 scFv used in the prior clinical trial, or a high affinity (HA) scFv generated by affinity maturation of the m971 scFv. SA- and HA-CD22 CAR T cells were evaluated in vitro and in vivo against clones of the pre-B ALL cell line, NALM6, which express CD22 at wild type levels (CD22WT), sub-physiologic levels (CD22Lo), supra-physiologic levels (CD22Hi) or in which CD22 was deleted (CD22Neg). We found that the amount of CD22 expressed on the leukemia cells resulted in dose-dependent expression of activation markers, such as CD69 and CD25 (p<0.05) on CD22 CAR T cells. Similarly, CAR T cell functions, such as the secretion of interferon-gamma (IFNg, p<0.0001) and interleukin-2 (IL-2, p<0.0001) as well as cytotoxic degranulation (p<0.0001) were all significantly impacted by the amount of CD22 on the surface of NALM6. A similar pattern of antigenic dose-response was seen in the signaling of CAR T cells, with phosphorylation of ERK reflecting the level of CD22 antigen (p<0.001) and correlating with the increased in vivo efficacy of the CAR T cells against CD22WT NALM6, relative to CD22Lo NALM6. Increasing the affinity of the CD22 CAR did not impact the in vivo efficacy against CD22WT NALM6 at either a therapeutic or subtherapeutic dose, however, HA-CD22 CAR T cells significantly prolonged the survival of NSG mice with CD22Lo NALM6, relative to SA-CD22 CAR T cells (p<0.01). The enhanced activity of HA-CD22 CAR T cells against CD22Lo leukemia did not correlate with improved in vitro functionality, as the HA-CD22 CAR T cells surprisingly demonstrated lower IL-2 secretion (p<0.01), lower proliferation (p<0.05) and diminished in vitro lysis of CD22Lo NALM6 (p<0.05), relative to SA-CD22 CAR T cells. ERK phosphorylation, however, was significantly increased in HA-CD22 CAR T cells (p<0.01) and was the only in vitro marker which correlated with the enhanced in vivo activity seen with the affinity-matured CAR. Previous clinical experience has demonstrated the importance of using a short linker (consisting of a single G4S sequence) between the heavy and light chains of the m971 scFv, therefore we next evaluated the impact of linker length on the activity of the HA-CD22 CAR. HA-CD22 CARs were generated with either a short- or long-linker (G4S x1 vs G4S x3, respectively) and evaluated in vitro and in vivo. While the short linker improved proliferation in vitro, there was no significant impact of linker length on cytokine production or lysis of CD22Lo NALM6. In a xenograft model, HA-CD22 CAR T cells with the long-linker demonstrated slower progression of CD22Lo leukemia and significantly prolonged survival of NSG mice with CD22WT leukemia relative to HA-CD22 CAR T cells with the short-linker (p<0.01). Taken together, these studies suggest that increasing the affinity of a scFv is a promising strategy for enhancing CAR sensitivity to low levels of target antigen, with the potential to decrease post-CAR T cell relapses due to antigen downregulation. Disclosures No relevant conflicts of interest to declare.

Mechanisms of cell death induced by arginase and asparaginase in precursor B-cell lymphoblasts

Arginase has therapeutic potential as a cytotoxic agent in some cancers, but this is unclear for precursor B acute lymphoblastic leukaemia (pre-B ALL), the commonest form of childhood leukaemia. We compared arginase cytotoxicity with asparaginase, currently used in pre-B ALL treatment, and characterised the forms of cell death induced in a pre-B ALL cell line 697. Arginase and asparaginase both efficiently killed 697 cells and mature B lymphoma cell line Ramos, but neither enzyme killed normal lymphocytes. Arginase depleted cellular arginine, and arginase-treated media induced cell death, blocked by addition of arginine or arginine-precursor citrulline. Asparaginase depleted both asparagine and glutamine, and asparaginase-treated media induced cell death, blocked by asparagine, but not glutamine. Both enzymes induced caspase cleavage and activation, chromatin condensation and phosphatidylserine exposure, indicating apoptosis. Both arginase- and asparaginase-induced death were blocked by caspase inhibitors, but with different sensitivities. BCL-2 overexpression inhibited arginase- and asparaginase-induced cell death, but did not prevent arginase-induced cytostasis, indicating a different mechanism of growth arrest. An autophagy inhibitor, chloroquine, had no effect on the cell death induced by arginase, but doubled the cell death induced by asparaginase. In conclusion, arginase causes death of lymphoblasts by arginine-depletion induced apoptosis, via mechanism distinct from asparaginase. Therapeutic implications for childhood ALL include: arginase might be used as treatment (but antagonised by dietary arginine and citrulline), chloroquine may enhance efficacy of asparaginase treatment, and partial resistance to arginase and asparaginase may develop by BCL-2 expression. Arginase or asparaginase might potentially be used to treat Burkitt lymphoma.

Neurokinin-1 receptor (NK1R) inhibition sensitizes APL cells to anti-tumor effect of arsenic trioxide via restriction of NF-κB axis: Shedding new light on resistance to Aprepitant.

While a batch of efforts are fastened on synthesizing the novel targeted anti-cancer agents, recent investigations have achieved a breakthrough in identifying a favorable anti-tumor activity for some supportive drugs, which their safety have been confirmed thus far. The results of the present study highlighted the efficacy of Aprepitant, an oral antagonist of the neurokinin-1 receptor (NK1R), against both APL (NB4) and pre-B ALL (Nalm-6) cell lines; however, a differential sensitivity pattern was found in these cells. To the best of our knowledge, this is the first time that the molecular mechanisms of resistance to Aprepitant have been investigated and, herein, we proposed that the effectiveness of Aprepitant could be overshadowed, at least partially, through over-activated nuclear factor-κB in Nalm-6 pre-B ALL cells. In contrast to Nalm-6, the cytotoxic property of Aprepitant in NB4 was divulged at the lower concentrations. Of particular interest, we found that the cytotoxicity of the inhibitor became even more evident in the synergistic experiments, where an enhanced reduction in viability was noted after treatment of NB4 cells with ATO-plus-Aprepitant. The stimulatory effect of NK1R inhibition on ATO cytotoxicity is probably mediated through up-regulation of p73, which can subsequently engage p21 and NF-κB pathway via transcriptional suppression of c-Myc. Taken together, the present study suggests that inhibition of NK1R using Aprepitant, either alone or in combination with chemotherapeutic drugs, could be a novel therapeutic strategy for the treatment of acute leukemia, especially APL, that may be clinically accessible in the near future.

The telomerase inhibitor MST-312 synergistically enhances the apoptotic effect of doxorubicin in pre-B acute lymphoblastic leukemia cells

BackgroundThere have been consistent efforts in concomitant administration of chemotherapeutic agents such as doxorubicin beside other more tumor-specific drugs in order to increase sensitivity of tumor cells to the cytotoxicity of doxorubicin and reduce necessary chemotherapeutic dosage. Telomerase-targeted therapy for cancer has received great attention because telomerase is expressed in almost all cancer cells but is inactive in most normal somatic cells. The present study was aimed to investigate the effect of telomerase inhibitor MST-312, a chemically modified derivative of epigallocatechin gallate (EGCG), on doxorubicin-induced apoptosis in pre-B acute lymphoblastic leukemia cells. Materials and methodsThe pre-B ALL cell lines (NALM-6 and REH) were cultured and treated with MST-312 and doxorubicin, separately and in combination. Cell viability was measured by trypan blue staining and MTT assay. Annexin-V/7-AAD staining by flow cytometry was used for evaluation of apoptosis. Gene expression of hTERT, c-Myc, Bax and Bcl-2, was detected by the quantitative Real-Time PCR. ResultsOur results showed that MST-312 exerted dose-dependent short-term cytotoxic and apoptotic effects on pre-B ALL cells. Combination of MST-312 and doxorubicin synergistically enhanced the cytotoxicity and apoptosis of both NALM-6 and REH cells. Consistently, apoptosis induction by the combined treatment was associated with significant reduction of Bcl-2, c-Myc and hTERT, and a marked increase of Bax mRNA expression levels, compared with the single treatments. ConclusionsOur data suggest that the synergistic anticancer effect induced by combination of MST-312 and doxorubicin represents a novel treatment strategy for pre-B ALL.

Centaurea albonitens extract enhances the therapeutic effects of Vincristine in leukemic cells by inducing apoptosis

Drug-induced toxicities and dose-related side effects are the major challenges in the conventional cancer therapy by the chemo drugs. On the other hand, herbal derivatives have obtained a great research interest in the field of therapeutic applications because of their more favorable specifications including less toxicity, cost-effective and more physiologically compatible than the chemical drugs. For this purpose, we evaluated methanolic extract prepared from Centaurea albonitens Turrill alone and in combination with Vincristine (VCR) for its potential cytotoxic effects in NALM-6, REH, NB4 and KMM-1 cell lines by using the various approaches. Centaurea genus is one of the current medicinal plants, which has used in traditional medicine, However, there are rare studies to examine its anticancer properties against hematologic malignant cells. In this study, we demonstrated Centaurea albonitens extract (CAE) induces cytotoxicity through G0/G1 phase arrest followed by apoptosis in a dose- and time- dependent manner, although with varying efficiency. Interestingly, normal cells didn’t exhibit significant cytotoxicity after CAE treatment. Moreover, we found that low dose of CAE enhances anti-cancer effects of VCR in pre-B ALL cell lines (NALM-6 and REH). Further investigations validated synergistic anticancer activities of VCR and CAE through inducing apoptosis without significant cell cycle arrest. Taken together, our results demonstrated for the first time that the methanolic extract of Centaurea albonitens can be considered as a potential anticancer agent and/or an enhancer of chemotherapeutic sensitivity of VCR.

Phosphatase of regenerating liver-3 is expressed in acute lymphoblastic leukemia and mediates leukemic cell adhesion, migration and drug resistance.

Phosphatase of regenerating liver-3 (PRL-3/PTP4A3) is upregulated in multiple cancers, including BCR-ABL1- and ETV6-RUNX-positive acute lymphoblastic leukemia (ALL). With this study, we aim to characterize the biological role of PRL-3 in B cell ALL (B-ALL). Here, we demonstrate that PRL-3 expression at mRNA and protein level was higher in B-ALL cells than in normal cells, as measured by qRT-PCR or flow cytometry. Further, we demonstrate that inhibition of PRL-3 using shRNA or a small molecular inhibitor reduced cell migration towards an SDF-1α gradient in the preB-ALL cell lines Reh and MHH-CALL-4. Knockdown of PRL-3 also reduced cell adhesion towards fibronectin in Reh cells. Mechanistically, PRL-3 mediated SDF-1α stimulated calcium release, and activated focal adhesion kinase (FAK) and Src, important effectors of migration and adhesion. Finally, PRL-3 expression made Reh cells more resistance to cytarabine treatment. In conclusion, the expression level of PRL-3 was higher in B-ALL cells than in normal cells. PRL-3 promoted adhesion, migration and resistance to cytarabine. PRL-3 may represent a novel target in the treatment of B-ALL.

Abstract 198: The phosphatase PRL-3 is expressed in primary B-ALL cells and is important for adhesion and migration

Abstract Introduction: Phosphatase of regenerating liver -3 (PRL-3) is a dual specificity phosphatase, and is associated with aggressiveness and metastatic disease in a number of solid tumors. Less is known about its role in hematological cancer, but we have previously shown that PRL-3 is expressed in primary multiple myeloma (MM) cells and MM cell lines. More recently PRL-3 is shown to be expressed in acute myeloid leukemia (AML), and associated with poor prognosis. Acute lymphoblastic leukemia (ALL) is the most common childhood cancer. Despite progress in treatment and survival, treatment related toxicity is a major concern. Therefore, it is a need for new clinical markers and therapy targets to optimize the treatment. We hypothesized that PRL-3 is expressed, and have oncogenic functions in B-ALL, and can be a possible clinical marker and a novel drug target. Methods: Primary ALL cells from peripheral blood were obtained from The Regional Biobank of Central Norway, and PRL-3 mRNA expression was measured with qRT-PCR. The PreB-ALL cell lines Reh and MHH-CALL-4 were used for further experiments. IL-7 (100 ng/mL) were used for stimulation. ShRNA against PRL-3 was used to knock down PRL-3 in Reh (shPRL-3) and for empty vector control (shMOCK) by use of lentiviral transduction. To study adhesion we used fibronectin (20 μg/mL) and stimulated with IL-7, IL-8 and IGF-1 (all 100 ng/mL) for 45 min in 37°C. Migration was studied with transwell permeable plates (pore size 3 μm) using SDF1α (75 ng/mL) as a chemoattractant. Further we examined the effect of the small molecular inhibitor PRL-3 Inhibitor I on ALL cell lines. Apoptosis and cell viability was determined by Annexin-FITC/-PI with flow cytometry, and by CellTiter-Glo® Luminescent Cell Viability assay kit, after 24-72 hours incubation with PRL-3 Inhibitor I. Results: 89% (16 of 18) of primary B-ALL samples expressed PRL-3, and 33% (6 of 18) at high levels. Stimulation with IL-7 induced expression of PRL-3 in a dose-dependent manner in Reh and MHH-CALL-4. The knock down of PRL-3 by shRNA was 80% efficient. Reh shMOCK cells were significantly more adherent to fibronectin after stimulation with IL-7, IL-8 and IGF-1 compared to Reh shPRL3. The cells’ ability to migrate towards a SDF1α gradient were significantly reduced in Reh shPRL-3 cells compared with Reh shMOCK. PRL-3 inhibitor I also reduced the viability and induced apoptosis in Reh cell line in a dose-dependent manner, but not in MHH-CALL-4 cell line, at a concentration of 40-80 μM. Conclusion: PRL-3 was expressed in 89% of primary B-ALL samples. Knock down of PRL-3 clearly reduced cell adhesion and migration. PRL-3 inhibitor I induced apoptosis and reduced cell viability in Reh, but not in MHH-CALL-4. This study indicates that PRL-3 could be important in the pathogenesis of B-ALL, and might be a suitable target for treatment in the future. Citation Format: Magnus Aassved A. Hjort, Pegah Abdollahi, Esten Vandsemb, Tobias Schmidt Slørdahl, Bendik Lund, Magne Børset, Torstein Baade Rø. The phosphatase PRL-3 is expressed in primary B-ALL cells and is important for adhesion and migration. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 198.

ROR1 Protein Expression Is Regulated By UHRF1 in t(1;19) Pre B-Cell ALL

Introduction: Acute lymphoblastic leukemia is the most common form of pediatric cancer, and a translocation between chromosomes 1 and 19 represents one of the most frequent cytogenetic abnormalities, accounting for ~5% of ALL cases. The 1;19 rearrangement results in a fusion between the E2A and PBX1 genes, and pre B-ALL cells carrying the E2A-PBX1 fusion are consistently arrested at an intermediate stage of B cell lineage development in which the cells express a functional pre B-cell receptor (pre-BCR). We have previously shown that receptor orphan tyrosine kinase receptor 1 (ROR1) is highly expressed on t(1;19) pre B-ALL blasts and required for their viability, even though ROR1 is not directly regulated by either the E2A-PBX1 fusion or the pre-BCR. Moreover, ROR1 exhibits compensatory signaling cross-talk with the pre-BCR such that maximal killing of t(1;19) pre B-ALL cells is achieved by combining silencing of ROR1 with siRNA and inhibition of the pre-BCR with kinase inhibitors such as dasatinib. However, clinical employment of this strategy is hampered as ROR1 is a pseudokinase whose biology is poorly understood and no ROR1 small-molecule inhibitors currently exist. Therefore, it is critical to advance our understanding of the upstream regulatory mechanisms responsible for ROR1 expression to uncover new therapeutic strategies for t(1;19) pre B-cell ALL.Methods: To identify potential regulators of ROR1 expression, we designed two siRNA panels targeting transcription factors predicted to bind the ROR1 locus or an array of chromatin remodeling factors and epigenetic regulators. Pre B-ALL cell lines that are positive or negative for the 1;19 rearrangement were screened with these siRNA panels and any gene targets required for the viability of t(1;19) pre B-cell ALL cells but not control cells lines were considered candidate regulators of ROR1 expression. Candidates were validated for the capacity to regulate ROR1 expression by qRT-PCR and immunoblot.Results: Screening of the t(1;19) pre B-cell ALL cell line, RCH-ACV, with our siRNA panels targeting transcription factors and chromatin remodeling/epigenetic regulators identified four potential regulators of ROR1 that were required for viability of RCH-ACV cells but not our negative control cell line, REH (t(12;21)-positive). Among these four candidates was UHRF1, which interacts with histone and DNA methyltransferases. Interestingly, silencing of UHRF1 by siRNA led to reduced ROR1 protein levels, but had no significant impact on ROR1 mRNA expression. Since one primary function of UHRF1 is to regulate DNA methylation, we hypothesized that UHRF1 regulates ROR1 protein expression through an intermediate gene whose transcription is impacted by UHRF1-mediated DNA methylation. To test this hypothesis, RCH-ACV cells were treated with the RNA synthesis inhibitor actinomycin D 48 hours after silencing UHRF1. This resulted in a transient rescue of ROR1 protein levels, indicating that UHRF1 regulation of ROR1 protein depends on the transcription of an intermediate gene whose gene product subsequently regulates ROR1 protein levels.Conclusions: Data from siRNA screens and subsequent validation experiments suggest that UHRF1 functions to regulate ROR1 protein levels, but not mRNA expression. This is intriguing due to the primary role of UHRF1 as a modulator of DNA and histone methylation. The observed rescue of ROR1 protein with actinomycin D suggests that UHRF1 regulation of ROR1 protein occurs through an undefined intermediary whose locus is methylated in a UHRF1-dependent manner and whose gene product modulates ROR1 protein levels. Inhibitors of additional stages of gene transcription, translation, and stabilization are currently being tested to continue characterizing UHRF1-dependent regulation of ROR1. Cumulatively, this work has identified a novel regulatory mechanism of ROR1 protein expression by UHRF1 in t(1;19) pre B-cell ALL. In addition to advancing our understanding of ROR1 biology, this regulatory mechanism suggests a new therapeutic strategy to target ROR1-expressing cancers. DisclosuresTyner:Incyte: Research Funding; Aptose Biosciences: Research Funding; Janssen Pharmaceuticals: Research Funding; Constellation Pharmaceuticals: Research Funding; Array Biopharma: Research Funding.

Dual Targeting of Ph+ ALL with Dasatinib and ABT-199 (Venetoclax)

Dual Targeting of Ph+ ALL with Dasatinib and ABT-199 (Venetoclax)

Co-Opting the Genetic Redundancy of the Pax Family to Treat PAX5 Mutated Pre-B ALL

Pre-B cell ALL is the most common pediatric malignancy. More than a third of cases acquire loss-of-function mutations of PAX5, a regulatory transcription factor which is required for the differentiation of B lymphocytes. Additionally, germline loss of function PAX5 mutation is a cause of familial pre-B cell ALL. These observations indicate that loss of PAX5 activity drives leukemogenesis. We have seen that PAX5 re-expression in PAX5 -mutated pre-B cell ALL restores differentiation capacity and promotes apoptosis. However, because the gene is deleted or otherwise mutated it may not be possible to easily repair or restore PAX5 expression in leukemic cells. Nevertheless, PAX5 possesses two closely related paralogs, PAX2 and PAX8, which are structurally and functionally similar to PAX5, yet are neither expressed in lymphocytes nor mutated in ALL. Toward the goal of exploiting PAX family genomic redundancy for therapeutic purposes, we have tested the ability of PAX2 and PAX8 to replace PAX5 in pre-B ALL cell lines. Preliminary results indicate that both PAX2 and PAX8 rescue B cell differentiation and function similarly to PAX5 in this context. These results include modulation of B cell developmental markers, CD10, CD19 and CD22, as well as induction of PAX5 target genes, CD79a and BACH2. Cells also display a reduction in size which may be indicative of the large to small B cell transition and importantly show increased levels of cell death following PAX2 and PAX8 expression. We have further investigated the epigenetic status of PAX2 and PAX8 promoters in B cells and find that both loci are hypermethylated, suggesting that demethylation with agents such as methotrexate, may represent a therapeutic entry point for activating expression of PAX5 paralogs. Finally, based on observations that electrical polarization during early embryogenesis regulates developmental transcription factor expression, including for PAX genes, we have tested the ability of approved drugs targeting ion channels for their ability to induce PAX factor expression and thereby complement for loss of PAX5 in pre-B cell ALL. The anti-parasitic drug ivermectin, which activates chloride channels, is one of several compounds that appear promising in early studies. DisclosuresNo relevant conflicts of interest to declare.

Novel CT domain-encoding splice forms of CTGF/CCN2 are expressed in B-lineage acute lymphoblastic leukaemia

IntroductionConnective tissue growth factor (CTGF/CCN2) has been shown previously to be aberrantly expressed in a high proportion of paediatric precursor B cell acute lymphoblastic leukaemia (pre-B ALL), suggesting a potential oncogenic role in this tumour type. We therefore assessed CTGF mRNA transcript diversity in B-lineage ALL using primary patient specimens and cell lines. MethodsCTGF mRNA expression was evaluated by quantitative real-time PCR and Northern blotting. We performed a structural analysis of CTGF mRNA by nested reverse-transcriptase PCR and examined CTGF protein diversity by immunoblotting. ResultsNorthern blot analysis of pre-B ALL cell lines revealed short CTGF transcripts that were expressed in association with the active phase of cellular growth. Structural analysis confirmed the synthesis of several novel CTGF mRNA isoforms in B-lineage ALL cell lines that were uniformly characterised by the retention of the coding sequence for the C-terminal (CT) domain. One of these novel spliceforms was expressed in a majority (70%) of primary pre-B ALL patient specimens positive for canonical CTGF mRNA. Evidence that these alternative transcripts have coding potential was provided by cryptic CTGF proteins of predicted size detected by immunoblotting. ConclusionThis study identifies for the first time alternative splicing of the CTGF gene and shows that a short CTGF splice variant associated with cell proliferation is expressed in most cases of primary CTGF-positive pre-B ALL. This novel variant encoding only the CT domain may play a role in pre-B ALL tumorigenesis and/or progression.

Loss of MXD3 induces apoptosis of Reh human precursor B acute lymphoblastic leukemia cells.

MXD3 is a transcription factor that plays an important role in proliferation of human DAOY medulloblastoma cells. Here, we demonstrate that MXD3 is highly enriched in human precursor B acute lymphoblastic leukemia (preB ALL) samples compared to mobilized peripheral blood mononuclear cells, bone marrow, or hematopoietic stem cells from healthy donors. MXD3 knock-down in the preB ALL cell line Reh resulted in decreased cell numbers with no change in G0/G1, S or G2/M populations but increased apoptosis compared to control cells. Our results suggest that MXD3 is important for survival of Reh preB ALL cells, possibly as an anti-apoptotic factor.

Efficacy of an Anti-CD22 Antibody-Monomethyl Auristatin E Conjugate in a Preclinical Xenograft Model of Precursor B Cell Acute Lymphoblastic Leukemia

Precursor B cell acute lymphoblastic leukemia (preB ALL) is the most common childhood cancer, as well as the leading cause of childhood cancer-related mortality. Despite overall progress in treatment, certain types of patients with preB ALL have a dismal prognosis with an overall survival of 30%. In addition, current approaches predispose these young patients to late effects, including secondary malignancies. Therefore, more efficacious and less toxic approaches are needed. Targeted therapy for leukemia has the potential to reduce off-target effects, thus minimizing toxicity and late effects and improving efficacy.Monoclonal antibodies (mAbs) have proven utility in leukemia therapy based on their ability to specifically target the leukemic clone and minimize off target effects. However, mAbs are generally not adequate as single agents because of limited efficacy. Antibody drug conjugates (ADCs) provide a method to deliver a potent toxin to the interior of antigen-positive tumor cells. CD22 is an ideal target for ADC-mediated therapeutics for B-cell malignancies because 1) there is high CD22 expression (more than 90%) in B-cell type ALL and 2) CD22 undergoes rapid internalization upon mAb binding. In this study, we evaluated the anti-CD22 (aCD22) mAb as a vehicle for the targeted delivery of Monomethyl Auristatin E (mMAE), a derivative of the cytotoxic tubulin modifier auristatin E. [Display omitted] First, we assessed the in vitro cytotoxicities of the aCD22 mAb-mMAE in preB ALL cell lines Reh and JM1. MTS assay showed that IC50 doses of aCD22 mAb-mMAE were 0.7nM and 1nM in Reh and JM1, respectively. Next, we assessed in vivo therapeutic efficacy of the aCD22 mAb-mMAE in a pre-clinical xenograft animal model of preB ALL, using a primary leukemia sample which was confirmed to be CD22 positive. Age matched female NOD/SCID/IL2Rg-/- (NSG) mice were randomly assigned to 4 treatment groups (n=8 per group): 1) PBS, 2) free mMAE (0.165mg/kg), 3) free aCD22 mAb (7.335mg/kg), and 4) aCD22 mAb-mMAE conjugate (7.5mg/kg). The dose of free aCD22 mAb and free mMAE was equivalent to those of each component in the aCD22 mAb-mMAE conjugate. Five million leukemia cells were inoculated per mouse via intra-bone marrow injection. Twenty four hours after leukemia inoculation, animals started receiving weekly iv treatments for 3 weeks. When compared to controls (PBS, free aCD22 mAb or mMAE treatments), the treatment with the aCD22 mAb-mMAE conjugate increased the median survival time of the mice by two fold (Figure. PBS vs. aCD22 mAb-mMAE p<0.005, free mMAE vs. aCD22 mAb-mMAE p<0.05, free aCD22 mAb vs. aCD22 mAb-mMAE p<0.05, by Gehan-Breslow-Wilcoxon test). Leukemia-related death was confirmed by necropsy. Harvested leukemia cells were assessed by flow cytometry and found to be HLA and CD22 positive. During treatment, the mice in all the treatment groups remained healthy and active, and did not lose weight. Toxicity was assessed with weekly CBC and chemistry panels which revealed no significant toxicity.In conclusion, we demonstrated that aCD22 mAb-mMAE is efficacious in a preclinical preB ALL xenograft mouse model. DisclosuresNo relevant conflicts of interest to declare.

Self-Enforcing Feedback Activation Between BCL6 and Tonic Pre-B Cell Receptor Signaling in Acute Lymphoblastic Leukemia

Background and hypothesis: Like mature B cell lymphoma, pre-B ALL originates from B cell precursors that critically depend on survival signals emanating from a functional (pre-) B cell receptor (BCR). While recent work successfully introduced BCR signaling inhibitors into patient care for various subtypes of mature B cell lymphoma, it is not known whether pre-BCR signaling represents a therapeutic target in pre-B ALL and in which cytogenetic subsets targeting of pre-BCR signaling will be effective. In this study we demonstrated that ALL can be subdivided into two groups that fundamentally differ with respect to pre-BCR signalling. We identified a novel mechanism of self-enforcing feedback activation between the transcription factor BCL6 and tonic pre-BCR signaling in pre-BCR+ ALL and proposed a dual targeting strategy of both BCL6 and pre-BCR related tyrosine kinases for the treatment of patients with pre-BCR+ ALL.Results: Flow cytometry analysis of surface pre-BCR expression (λ5, VpreB), cytoplasmic μ heavy chain (μHC) expression and intracellular Ca2+ signal in 29 patient-derived pre-B ALL xenograft samples and cell lines showed pre-BCR expression and activity in a subset of pre-B ALL, including all TCF3-PBX1 cases studied (n=4) and two cases with deletions at 6q21. Studying 830 pre-B ALL cases from four clinical trials (MDACC, St. Jude, COG P9906 and ECOG E2993), tonic pre-BCR signaling and constitutive PI3K-AKT activation was found in 112 cases (13.5%), including 93% TCF3-PBX1 (53 of 57), del (6)(q21) (7 of 7), PBX1 (1q23) duplication (4 of 4), MLL-rearrangement (3 of 86), hyperdiploid (2 of 43) and other (43 of 406) pre-B ALL cases. In other major ALL subtypes, we found no evidence for pre-BCR expression and activity, including BCR-ABL1 (0 of 196) and ETV6-RUNX1 (0 of 31). We found frequent 1q23 (PBX1) duplication, TCF3-PBX1 or other PBX1-rearrangement, 6q21 (PRDM1) deletion in ALL cells with tonic pre-BCR signaling.Development of a genetic mouse model for inducible ablation of Bcl6. Pre-BCR-induced activation of BCL6 relieves PRDM1-mediated repression of pre-BCR signaling components and positively regulates pre-BCR signaling output at the transcriptional level. The clinical data (COG P9906, ECOG E2993) revealed that high mRNA levels of BCL6 at the time of diagnosis is predictive of poor clinical outcome specifically in patients with pre-BCR+ ALL but not ALL cells lacking pre-BCR expression. These findings suggest an important role of BCL6 as a cofactor of pre-BCR signaling in a large subset of ALL. To directly test the role of Bcl6- and pre-BCR interactions, we generated a novel mouse model for inducible Cre-mediated deletion of Bcl6 exons 5-10, flanked by loxP sites. For lineage-specific deletion in vivo, we crossed these mice with an Mb1-Cre deleter strain, in which Bcl6 was deleted in pro-B cells, resulting in a differentiation block at the pre-B cell stage. Deletion of Bcl6 in mouse pre-BCR+ ALL and expression of a dominate-negative form of BCL6 in human primary pre-BCR+ALL cells, both rapidly induced cell death, indicating BCL6 cooperates with the pre-BCR in leukemic transformation.Cooperation between pre-BCR and BCL6 signaling. Inhibition of BCL6 via the specific BCL6 inhibitor RI-BPI showed compromised colony formation and induced cell cycle arrest. Interestingly, constitutive BCL6 expression was sensitive to inhibition of SYK and SRC tyrosine kinases downstream of the pre-BCR. Treating 6 pre-BCR+ and 8 pre-BCR- patient-derived ALL samples with the SYK inhibitor (PRT06207), BTK inhibitor (Ibrutinib) or a broader SRC and BTK inhibitor Dasatinib, we observed remarkably decreased BCL6 expression and increased apoptosis in pre-BCR+ but not pre-BCR- ALL cells. In vivo treatments with Dasatinib prevented leukemia initiation and significantly prolonged survival of the recipient mice that were injected with primary pre-BCR+ ALL cells, compared to non-treatment or Nilotinib-treatment. These data demonstrate that both inhibition of BCL6 and pre-BCR signaling selectively killed patient-derived pre-BCR+ ALL cells.Conclusions: Our study identified two distinct subtypes of pre-B ALL that fundamentally differ with respect to pre-BCR signaling. Tonic pre-BCR signaling engages a BCL6-dependent, self-enforcing amplification loop. Based on these findings, we propose a dual targeting strategy of BCL6 and pre-BCR tyrosine kinases for the treatment of patients with pre-BCR+ALL. DisclosuresNo relevant conflicts of interest to declare.

NF-kappa B mediated Up-regulation of CCCTC-binding factor in pediatric acute lymphoblastic leukemia

BackgroundAcute lymphoblastic leukemia (ALL) is the most frequently occurring malignant neoplasm in children. Despite advances in treatment and outcomes for ALL patients, the pathogenesis of the disease remains unclear. Microarray analysis of samples from 100 Chinese children with ALL revealed the up-regulation of CTCF (CCCTC binding factor). CTCF is a highly conserved 11-zinc finger protein that is involved in many human cancers; however, the biological function of CTCF in pediatric ALL is unknown.MethodsThe expression patterns of CTCF were evaluated in matched newly diagnosed (ND), complete remission (CR), and relapsed (RE) bone marrow samples from 28 patients. The potential oncogenic mechanism of CTCF and related pathways in leukemogenesis were investigated in leukemia cell lines.ResultsWe identified significant up-regulation of CTCF in the ND samples. Importantly, the expression of CTCF returned to normal levels after CR but rebounded in the RE samples. In the pre-B ALL cell line Nalm-6, siRNA-mediated silencing of CTCF expression promoted cell apoptosis and reduced cell proliferation; accordingly, over-expression of a cDNA encoding full-length CTCF protected cells from apoptosis and enhanced cell proliferation. Furthermore, inhibition or activation of the nuclear factor-kappa B (NF-κB) pathway resulted in marked variations in the levels of CTCF mRNA and protein in leukemic cells, indicating that CTCF may be involved downstream of the NF-κB pathway. Moreover, inhibition of the NF-κB pathway increased cell apoptosis, which was partially rescued by ectopic over-expression of CTCF, suggesting that CTCF may play a significant role in the anti-apoptotic pathway mediated by NF-κB.ConclusionsOur results indicate that CTCF serves as both an anti-apoptotic factor and a proliferative factor in leukemic cells. It potentially contributes to leukemogenesis through the NF-κB pathway in pediatric ALL patients.