REH Cells Research Articles

Sulfurtransferases and Cystathionine Beta-Synthase Expression in Different Human Leukemia Cell Lines.

The studies concerned the expression of sulfurtransferases and cystathionine beta-synthase in six human leukemia cell lines: B cell acute lymphoblastic leukemia-B-ALL (REH cells), T cell acute lymphoblastic leukemia-T-ALL (DND-41 and MOLT-4 cells), acute myeloid leukemia—AML (MV4-11 and MOLM-14 cells), and chronic myeloid leukemia—CML (K562 cells). Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis were performed to determine the expression of thiosulfate sulfurtransferase, 3-mercaptopyruvate sulfurtransferase, gamma-cystathionase, and cystathionine beta-synthase on the mRNA and protein level. Interestingly, we found significant differences in the mRNA and protein levels of sulfurtransferases and cystathionine beta-synthase in the studied leukemia cells. The obtained results may contribute to elucidating the significance of the differences between the studied cells in the field of sulfur compound metabolism and finding new promising ways to inhibit the proliferation of various types of leukemic cells by modulating the activity of sulfurtransferases, cystathionine beta-synthase, and, consequently, the change of intracellular level of sulfane sulfur as well as H2S and reactive oxygen species production.

The Correlation Between Children’s Acute Lymphoblastic Leukemia Drug Resistance System Induced by Metabolomics-Based 6-Mercaptopurine and Hypoxanthine-Guanine Phosphoribosyl Transferase 1 Protein

This study aimed to explore 6-mercaptopurine (MP)-induced children’s acute lymphoblastic leukemia (ALL) drug resistance system and leukemia hypoxanthine-guanine phosphoribosyl transferase 1 (HPRT1) protein. Based on metabonomics, drug resistance of 6MP-Reh cell line was established by increasing concentration administration method, and the degree of drug resistance of 6MP-Reh was verified by apoptosis test, western blotting (WB) test, and drug sensitivity test. The changes of tissue inhibitor of matrix metalloproteinase (TIMP) and thioguanosine monophosphate (TGMP) in drug-resistant cells were detected through liquid chromatograph (LC)/mass spectrometer (MS). The 6MP-Reh-wt cell line was established by lentivirus infection, so as to verify the correlation between HPRT1 and drug resistance mechanism. The results showed that the inhibition concentration (IC50) value, cell vitality (CV), apoptosis rate, and 6-MP content of 6MP-Reh were higher hugely than those of Reh (P < 0.05). The contents of HPRT1, TIMP, and TGMP in 6MP-Reh cells were lower sharply than the contents of Reh cells (P < 0.001). The IC50 value of 6MP-Reh-wt was also lower steeply than the value of 6MP-Reh (P < 0.001), and the concentrations of TIMP and TGMP increased obviously (P < 0.05). Therefore, it indicated that the mutation of HPRT1 in drugresistant cell lines could lead to a decrease in their viability and cause leukemia cells to develop resistance to 6-MP. In addition, HPRT1 gene could improve their resistance to 6-MP.

PON2 blockade overcomes dexamethasone resistance in acute lymphoblastic leukemia

ABSTRACT Objectives The high frequency of chemotherapy resistance is ultimately responsible for clinical relapse in acute lymphoblastic leukemia (ALL). Nevertheless, the molecular mechanism relevant to glucocorticoid (GC) resistance remains ambiguous. Methods Quantitative real-time polymerase chain reaction and Western blot were performed to detect the expressions of paraoxonase 2 (PON2), Bcl-2 and Bax. shRNA was used to knockdown PON2 expression in SUP-B15 and REH cell. CCK-8 and flow cytometry assay were conducted to monitor the changes of proliferation and apoptosis in ALL cells. The growth of ALL REH cells in vivo was determined using transplanted tumor model. Results This study was designed to identify GC resistance-associated genes by means of the transcriptome chip from the public Gene Expression Omnibus database, and preliminarily investigation of dexamethasone (DEX)-resistance mechanism in ALL. We disclosed that PON2 expression was elevated in ALL patients and especially higher in DEX-resistance ALL patients. Then, cell apoptosis assay suggested that silencing of PON2 dramatically promoted in DEX-resistant ALL cells apoptosis and the activity of Caspase 3 induced by DEX administration. In xenograft tumor model, PON2 knockdown significantly reduced DEX-resistant ALL cells growth in immunodeficient mice. Conclusions Collectively, inhibition of PON2 may represent a novel method to restore the sensitivity of treatment-resistant ALL to GC-induced cell death.

Autophagy Regulates VDAC3 Ubiquitination by FBXW7 to Promote Erastin-Induced Ferroptosis in Acute Lymphoblastic Leukemia.

Background: The discovery of ferroptosis is a major breakthrough in the development of cancer treatments. However, the mechanism by which ferroptosis contributes to acute lymphoblastic leukemia (ALL) is to be clarified. Here, we explored erastin-induced ferroptosis in ALL cells and the impact of autophagic activity on this process. Materials and Methods: Cell viability was evaluated in various ALL cell lines following erastin treatment by the MTS assay, while cell death was evaluated via a trypan blue assay. Immunoblotting and quantitative real-time PCR were used to detect protein and mRNA expression, respectively. The UbiBrowser database was used to predict the E3 ligase of VDAC3, which was confirmed by immunoprecipitation. The role of FBXW7 in erastin-induced ferroptosis in vitro was evaluated via lentiviral-mediated silencing and overexpression. ALL xenograft mice were used to observe the impact of autophagy on erastin-induced ferroptosis. Results: Resistance to erastin-induced ferroptosis was higher in Jurkat and CCRF-CEM cells than in Reh cells. The sensitivity could be modified by the autophagy activator rapamycin (Rapa) and the autophagy inhibitor chloroquine (CQ). Rapa sensitized ALL cells to erastin-induced ferroptosis. In ALL xenograft mice, the combination treatment of Rapa and erastin resulted in longer survival time than those observed with erastin or Rapa treatment alone. VDAC3 was regulated by autophagy post-transcriptionally, mainly via the ubiquitin-proteasome system (UPS). FBXW7 was verified as a specific E3 ligase of VDAC3. FBXW7 knockdown attenuated VDAC3 degradation by suppressing its ubiquitination, thereby increasing the sensitivity of ALL cells to erastin. Conclusion: Autophagy regulated erastin-induced ferroptosis via the FBXW7-VDAC3 axis. Rapa sensitized ALL cells to erastin-induced ferroptosis both in vitro and in vivo. Our findings provide potential therapeutic targets for ALL.

Glucocorticoid-Resistant B-Cell Acute Lymphoblastic Leukemic Cells Can be Targeted By BCR-Signaling Inhibition

Glucocorticoids (GCs) remain a backbone component of therapeutic regimens for childhood B-cell acute lymphoblastic leukemia (B-ALL). GCs resistance is a strong prognostic marker of relapse making its understanding an important challenge to be addressed to improve overall patients outcome.Healthy B-cell development is characterized by checkpoints where critical regulatory signaling influences the fate of developing B-cells. These stages are vulnerable for leukemic transformation and as we previously demonstrated, the developmental and functional state of B-ALL cells are of critical importance in treatment failure. Using this developmental framework, we examined the effect of GCs on healthy and malignant B-cells to better understand mediators of GC resistance and ways to overcome it.To define the dynamics of the transcriptional networks surrounding B-cell developmental checkpoints, we performed transcriptomic analysis of sorted pre-proB, pro-B and pre-B cells from 3 healthy donors. We found a coordinated upregulation of B cell receptor (BCR) and Glucocorticoid Receptor (GCR) pathways in healthy B cells during their development. Single cell proteomic analysis of these healthy populations confirmed the coordinated expression of GCR with early B-cell phenotypic markers. Furthermore, in vitro treatment of healthy B-cells with the GC dexamethasone (dex) demonstrated cycling pro-B/pre-B cells to be the most sensitive to GC-induced cell death.Given the importance of GCs in B-ALL treatment, we investigated GCs effects and resistance in NALM6 and REH cell lines. NALM6, expressing high levels of GCR, were significantly sensitive to dex treatment in terms of cell cycle arrest and cell death. By contrast, REH cells were resistant to dex treatment as they lack the GCR. Retroviral transfection of NR3C1 (GCR gene) in REH cells (REH GCR) resulted in acquired sensitivity to dex. To explore the potential crosstalk between GCR and BCR pathways, we also treated cells with BCR signaling inhibitor, dasatinib (das), alone or in combination with dex. While both cell lines survived to treatment with das, the combined treatment increased apoptosis compared to dex alone in NALM6 cells (p=0.0179) and REH GCR cells (p=ns). Whole transcriptome sequencing of dex-resistant cells revealed upregulation of BCR downstream signaling as one of the main pathways associated with resistance.The cell line data implicated active BCR signaling as a path to GCs resistance, so we next analyzed 19 B-ALL primary samples by mass cytometry, after in vitro exposure for 48 hrs to same treatments. Across the entire cohort, dex induced a significant reduction in viability (p=0.0007), compared to vehicle. Treatment with das and dex+das also decreased cell viability compared to vehicle (p=0.0038 and p=0.0002) although was not significant to dex alone. Interestingly dex-resistant cells showed a phenotypic modulation compatible with a late pre-B phenotype with increased CD45 and CD20 expression. In addition, surviving cells showed an activation of downstream targets of BCR signaling such as pSYK, pRPS6 and pCREB that was partially blunted by dasatinib treatment.To understand whether the phenotype and signaling modulation induced by dexamethasone also occurs in patients after treatment with GCs, we analyzed minimal residual disease (MRD) cells following 8 days of treatment with GCs from 9 B-ALL patients. This analysis confirmed our previous findings with MRD cells having same late pre-B cells phenotype and signaling profile as in vitro treated cells.Finally, we tested whether targeting of pre-BCR signaling via dasatinib, could overcome resistance in vivo. We evaluated engraftment of luciferase-expressing NALM6 cells at different timepoints after tail vein injection in mice treated with vehicle, dex, das or dex + das. Bioluminescence analysis revealed a significant reduction of early and late engraftment in the dex + das group compared to vehicle-treated mice. Furthermore, mice receiving the combined treatment also experienced a significant survival advantage as assessed by log rank test (p=0.0002).Taken together these data suggest a coordinated interplay between BCR and GCR pathways in healthy and leukemic B cells. GCs-resistant leukemic cells showed a mature pre-B phenotype that is vulnerable to BCR signaling inhibition in vitro and in vivo suggesting new therapeutic options to overcome GC resistance in childhood B-ALL. DisclosuresBiondi: Bluebird: Other: Advisory Board; Novartis: Honoraria; Incyte: Consultancy, Other: Advisory Board; Amgen: Honoraria; Colmmune: Honoraria. Bava: 10x Genomics: Current Employment. Davis: Jazz Pharmaceuticals: Research Funding; Novartis Pharmaceuticals: Honoraria.

Pre-Clinical Evaluation of Novel L-Asparaginase Mutants for the Treatment of Acute Lymphoblastic Leukemia

IntroductionAcute Lymphoblastic Leukemia (ALL) accounts for 20% of all hematological malignancies. L-Asparaginase has been a mainstay of ALL for the last 6 decades and is also included in the WHO list of essential medicines for ALL. Escherichia coli L-asparaginase (EcA) was the first asparaginase to be approved for clinical use. However being isolated from bacteria, EcA has many side-effects which in turn affects the tolerability and efficacy of the drug. EcA administration may cause strong immunogenic and hypersensitive reactions in the patients, necessitating withdrawal of the drug. Sensitive individuals react to repeated EcA administration with formation of anti-drug antibodies (ADAs) that bind to and inactivate the enzyme leading to inadequate plasma levels of EcA. Another serious drawback of EcA is the glutaminase activity which leads to neurotoxicity. Other side effects include hepatotoxicity, thromboembolism and pancreatitis. Although a number of attempts have been made to alleviate these problems by rational protein engineering, the optimization of therapy with EcA for ALL patients still remains a challenge. In an attempt to deal with these problems, we created several EcA mutants. On the basis of their activity, stability and antigenicity we short-listed four EcA mutants (Mutant A, B, C and D) having favourable properties for further development.MethodsWe identified and mutated several B-cell epitopes and amino acid residues at the EcA interface that are responsible for activity, stability and antigenicity. Enzyme activity was measured at 37 oC (optimum temperature for EcA). Glutaminase activity of the mutants was measured and compared to the wild type EcA. The cytotoxicity of the EcA variants was verified in ALL sensitive REH cell lines by performing MTT assay after 24 h incubation. Further the antigenicity of the mutants was assessed by performing indirect ELISA where the binding of the mutants to the commercially available l-asparaginase antibody was analysed. Further, in vivo immunogenicity was evaluated by immunizing Balc C mice with primary and booster doses of EcA mutants over 66 days followed by the measurement of IgG and IgM titers. In addition, the binding of wild-type EcA and mutants to pre-existing anti-asparaginase antibodies in serum isolated from primary and relapsed ALL patients receiving asparaginase therapy was studied by indirect ELISA. Pharmacokinetics of the mutants was evaluated in female Balb C mice by plotting the asparaginase activity-time curve till 24 h following administration of a single i.v. dose of 50 IU/kg and compared with the wildtype. Finally the safety of the EcA mutants was determined by performing single-dose acute toxicity study at 3 dose levels in Balb C mice.ResultsAt 37 oC, we did not find any significant difference in asparaginase activity of any EcA variant with the wild-type. All four variants showed markedly reduced glutaminase activity as compared to wild-type EcA (P<0.05). In MTT assay Mutant D showed 34.02%, Mutant B (32.4%), Mutant C (31.4%), Mutant A (24.22%), and wild type EcA (24.37%) reduction in REH cell viability in comparison to untreated cells. Binding to commercially available anti-asparaginase antibody was 49.09%, 32.63%, 27.43% less for Mutant D, Mutant B and Mutant C respectively compared to wild type EcA. Mice immunized with Mutant D showed 5-fold lower titres of IgG and 4-fold lower titres of IgM in comparison to wild type. Similarly, when compared to wild type, mice immunized with Mutant C showed 2.5-fold lower titres of IgG and 3.5-fold lower titres of IgM. At the same time Mutants B, C and D showed 2-3 fold less binding to pre-existing anti-asparaginase antibodies in samples collected from primary ALL patients undergoing asparaginase therapy. Similarly mutants B, C and D showed approximately 2-fold less binding to pre-existing anti-asparaginase antibodies in samples collected from relapsed ALL patients. Pharmacokinetic profiling showed that half life of Mutant A (267.28 ± 9.74), Mutant B (213.29 ± 6.53) and Mutant D (273.83 ± 35.45) was significantly longer than the wild type (102.17 ± 7.7). In acute toxicity study, we did not observe any significant toxicity of the mutants over the wildtype EcA. The findings are summarized in the figure.ConclusionConsidering the immunogenicity, antigenicity and pharmacokinetics, mutant D emerged as a potent drug candidate for further development in the treatment of ALL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Piggybac Transposon Mediated CD19 CAR-T Cells Derived from CD45RA-Positive PBMCs Possess Potent and Sustained Antileukemic Function

Background: The quality of chimeric antigen receptor (CAR)-T cell products, including the expression of memory and exhaustion markers, has been shown to influence their long-term functionality. We previously demonstrated that piggyBac (PB) transposon-mediated CD19 CAR-T cells exhibit memory-rich phenotype that is characterized by a high proportion of CD45RA+/CCR7+ T cell fraction. To further investigate the favorable phenotype of PB-CD19 CAR-T cells, we generated PB-CD19 CAR-T cells from CD45RA+ and CD45RA− peripheral blood mononuclear cells (PBMCs) (RA+ CAR and RA− CAR, respectively), and compared their phenotype and antitumor function.Methods: CD45RA+ and CD45RA− PBMCs were isolated by magnetic selection from whole PBMCs, then the CD19-CAR transgene was transduced into these cells using the PB transposon system, as described previously. Transduction efficiency of CD19 CAR transgene was determined 24 hours by flow cytometry after transduction. The phenotype of CD19 CAR-T was evaluated by flow cytometry on day 14. High throughput RNA sequencing was performed to see the T cell activation/exhaustion profile upon antigen stimulation. Sequential killing assays were performed by adding fresh tumor cells into CAR-T cells co-cultured with tumor cells every three days by restoring an effector target ratio of 1:1. To see the durable antitumor efficacy in vivo, we performed in vivo stress test, in which CAR T-cells dosage was lowered to the functional limits, so that these CAR-T cells should be maintained and expanded in vivo, to achieve the antitumor efficacy. We injected 5 x 10 5 of firefly luciferase-labeled CD19+ tumor cells (REH) into NSG mice via tail vein, then these mice were treated with 1 x 10 5 of CD19 RA+ CAR-T, RA− CAR-T, or control CAR-T cells, respectively, at day 6 after the tumor injection.Results: RA+ CAR T cells demonstrated better transient transduction efficiency 24 h after transduction (RA+ CAR-T: 77.5 ± 9.8% vs RA− CAR-T: 39.7 ± 3.8%), and superior expansion capacity after 14 days of culture than RA− CAR-T cells (RA+ CAR-T: 32.5 ± 9.3-fold vs RA− CAR-T: 11.1 ± 5.4-fold). RA+ CAR-T cells exhibited dominant CD8 expression (RA+ CAR-T: 84.0 ± 3.4% vs RA− CAR-T: 34.1 ± 10.6%), less expression of exhaustion marker PD-1 (RA+ CAR-T: 3.1 ± 2.5% vs RA− CAR-T: 19.2 ± 6.4%) and T cell senescence marker CD57 (RA+ CAR-T: 6.8 ± 3.6% vs RA− CAR-T: 20.2 ± 6.9%), and enrichment of naïve/stem cell memory fraction (CAR+/CD45RA+CCR7+ fraction; RA+ CAR-T: 71.9 ± 9.7% vs RA− CAR-T: 8.0 ± 5.3%), which were associated with longevity of CAR-T cells. Transcriptome analysis revealed that RA+ CAR-T cells exhibited the enrichment of naïve/memory phenotype and less expression of canonical exhaustion markers, and these exhaustion profiles even maintained after the antigen stimulation. RA+ CAR-T cells demonstrated sustained killing activity even after multiple tumor rechallenges in vitro, without inducing exhaustion marker expression of PD-1. Although antigen stimulation could increase CAR expression, leading to tonic CAR signaling and exhaustion, in our study, the expression of CAR molecule on the cell surface following antigen stimulation in RA+ CAR was controlled at a relatively lower level that in RA− CAR-T cells. RA+ CAR-T cells achieved prolonged tumor control with expansion of CAR-T cells than RA− CAR-T cells in in vivo stress test (Fig.1A-C). On day15, bone marrow studies in RA+ CAR group exhibited abundant human CD3 positive T cells with less expression of PD-1, and relatively smaller amount of REH cells than RA− CAR group (Fig.1D). Furthermore, in two of long-lived mice in RA+ CAR group, human CD3 positive T cells were expanded even day 50 after treatment as confirmed by sequential bone marrow studies (Fig.1E), which indicated the antigen-induced proliferation and long-term functionality of RA+ CAR-T cells in vivo.Conclusion: Our results suggest that PB-mediated RA+ CAR-T cells exhibit memory-rich phenotype and superior antitumor function, thereby indicating the usefulness of CD45RA+ PBMC as a starting material of PB-CAR-T cells. [Display omitted] DisclosuresYagyu: AGC Inc.: Research Funding. Nagao: AGC Inc.: Current Employment. Kubota: AGC Inc.: Current Employment. Shimizu: AGC Inc.: Current Employment. Nakazawa: AGC Inc.: Research Funding; Toshiba Corporation: Research Funding.

Synergism between IGF2BP1 and ETV6-RUNX1 in the Pathogenesis of ETV6-RUNX1 Positive B-Acute Lymphoblastic Leukaemia

Acute lymphoblastic leukaemia (ALL) is the most common pediatric haematological malignancy with a prevalence of around 85% cases of B cell origin. ETV6-RUNX1 is the most common genetic aberration in childhood B-ALL with a prevalence of around 25% in Western countries and a lower prevalence in Asian countries. ETV6-RUNX1 presence portends a good prognosis in B-ALL. ETV6-RUNX1 translocation has been observed in 1-5% of healthy newborns, indicating that it is not sufficient for leukemogenesis, and the need to accumulate secondary mutations for leukemogenesis.We have previously demonstrated the specific overexpression of Insulin like Growth Factor 2 Binding Protein 1 (IGF2BP1), an oncofetal RNA binding protein, in ETV6-RUNX1 positive B-ALL. Overexpression of the ETV6-RUNX1 fusion protein in 7OZ/3, a murine B-ALL cell line increased endogenous Igf2bp1 levels in a dose dependent manner.Conversely, knockout of IGF2BP1 in Reh-Cas9-GFP expressing B-ALL cell line, a modified version of Reh (cell line positive for ETV6-RUNX1 translocation), using 2 different guide RNAs led to a decrease in the ETV6-RUNX1 transcript expression in the IGF2BP1 KO Reh-Cas9 cells. This was accompanied by a decrease in cell proliferation and increased chemosensitivity to prednisolone.A similar phenotype was observed in Reh-Cas9 cells after knockout of ETV6-RUNX1 fusion transcript hinting towards a synergism between the two proteins in tumour cell survival and chemosensitivity. Interestingly, there appears to be a positive correlation between prednisolone resistance and ETV6-RUNX1 positivity in our patient cohort too, thus supporting the in vitro finding. Previous studies have shown the binding of IGF2BP1 to the ETV6-RUNX1 fusion transcript which was also confirmed by us.To study the in vivo synergistic effect between ETV6-RUNX1 fusion protein and IGF2BP1, we used a murine bone marrow transplant model. The individual genes were overexpressed alone (IGF2BP1/ETV6-RUNX1) or together (IGF2BP1+ETV6-RUNX1) in separate groups of lethally irradiated mice (n=8 per group). In the group where both the genes were overexpressed together, there was clonal expansion and hypercellularity in the bone marrow. This was accompanied by an increase in the number of lineage negative cells (Lin-), progenitors including HSCs, LMPPs and c-kit+ cells which also showed high Ki67 positivity. The marrow hypercellularity led to extramedullary haematopoiesis which was seen as significant splenomegaly.In addition, analysis of the endogenous levels of Igf2bp1 in the bone marrow of the primary murine samples belonging to the ETV6-RUNX1 fusion protein overexpression group resulted in an increase in its levels, thus confirming our in vitro finding suggesting its induction by ETV6-RUNX1 fusion protein.RNA-Seq of IGF2BP1 KO cells identified enrichment of targets of the TNF alpha/NFκB signalling and K-Ras pathways hinting towards their dependency on IGF2BP1. The direct targets of IGF2BP1 were identified by RIP-Seq (RNA-Immunoprecipitation-Seq) analysis in Reh cell line. Numerous genes from the oncogenic signalling pathways including the TNF alpha/ NFκB signalling pathway were identified as IGF2BP1 targets. We validated some overexpressed genes from the TNF alpha/NFκB signalling and PI3K pathway in primary patient samples. We found significant overexpression of some of the genes (IL6ST, NFAT5, CDK6, MDM2, CCND1, NGFR) belonging to these pathways in the ETV6-RUNX1 positive group (n=39) compared to the negative cohort (n=111) as well as MACS sorted CD19+ B-cells (n=5). The decrease in the ETV6-RUNX1 transcript levels after downregulation of IGF2BP1 and overlap between the oncogenic pathways clearly shows an interdependency between the two genes.Overall, our results suggest a potential feedback mechanism between ETV6-RUNX1 fusion protein and IGF2BP1 in the pathogenesis of leukemia development and IGF2BP1 can be utilised as an ideal therapeutic target for this particular subtype. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

DNA crosslinking and recombination-activating genes 1/2 (RAG1/2) are required for oncogenic splicing in acute lymphoblastic leukemia.

BackgroundAbnormal alternative splicing is frequently associated with carcinogenesis. In B‐cell acute lymphoblastic leukemia (B‐ALL), double homeobox 4 fused with immunoglobulin heavy chain (DUX4/IGH) can lead to the aberrant production of E‐26 transformation‐specific family related gene abnormal transcript (ERGalt) and other splicing variants. However, the molecular mechanism underpinning this process remains elusive. Here, we aimed to know how DUX4/IGH triggers abnormal splicing in leukemia.MethodsThe differential intron retention analysis was conducted to identify novel DUX4/IGH‐driven splicing in B‐ALL patients. X‐ray crystallography, small angle X‐ray scattering (SAXS), and analytical ultracentrifugation were used to investigate how DUX4/IGH recognize double DUX4 responsive element (DRE)‐DRE sites. The ERGalt biogenesis and B‐cell differentiation assays were performed to characterize the DUX4/IGH crosslinking activity. To check whether recombination‐activating gene 1/2 (RAG1/2) was required for DUX4/IGH‐driven splicing, the proximity ligation assay, co‐immunoprecipitation, mammalian two hybrid characterizations, in vitro RAG1/2 cleavage, and shRNA knock‐down assays were performed.ResultsWe reported previously unrecognized intron retention events in C‐type lectin domain family 12, member A abnormal transcript (CLEC12Aalt) and chromosome 6 open reading frame 89 abnormal transcript (C6orf89alt), where also harbored repetitive DRE‐DRE sites. Supportively, X‐ray crystallography and SAXS characterization revealed that DUX4 homeobox domain (HD)1‐HD2 might dimerize into a dumbbell‐shape trans configuration to crosslink two adjacent DRE sites. Impaired DUX4/IGH‐mediated crosslinking abolishes ERGalt, CLEC12Aalt, and C6orf89alt biogenesis, resulting in marked alleviation of its inhibitory effect on B‐cell differentiation. Furthermore, we also observed a rare RAG1/2‐mediated recombination signal sequence‐like DNA edition in DUX4/IGH target genes. Supportively, shRNA knock‐down of RAG1/2 in leukemic Reh cells consistently impaired the biogenesis of ERGalt, CLEC12Aalt, and C6orf89alt.ConclusionsAll these results suggest that DUX4/IGH‐driven DNA crosslinking is required for RAG1/2 recruitment onto the double tandem DRE‐DRE sites, catalyzing V(D)J‐like recombination and oncogenic splicing in acute lymphoblastic leukemia.

Autophagy Regulates the Sensitivity of Acute Lymphoblastic Leukemia Cells to Ferroptosis Activator by Influencing Iron Homeostasis

To investigate the effect of autophagy to the ferroptosis in acute lymphocytic leukemia (ALL) cells and its mechanism. ALL cell lines (including Reh, Jurkat and CCRF-CEM) were selected. The cell viability was detected by MTS assay and trypan blue staining was used to evaluate the death of the cell. The expression of autophagy related protein (including p62, LC3I/II) and Ferritin in ALL cells were detected by Western blot. The alteration of labile iron pool (LIP) in ALL cells was evaluated by flow cytometry. Reh cells showed sensitivity to ferroptosis activator Erastin, while Jurkat and CCRF-CEM cells showed resistant. Autophagy activator rapamycin could promote the sensitivity of Jurkat and CCRF-CEM cells to Erastin, and the ferroptosis of the cells (P<0.001). Autophagy inhibitor chloroquine could reduce the sensitivity of Reh cells to Erastin and resist the ferroptosis of the cells (P<0.001). The expression of Ferritin could be down-regulated after autophagy was activated in Jurkat and CCRF-CEM cells (P<0.05), while the level of LIP was significantly increased (P<0.05). Inhibiting the autophgy in Reh cells could up-regulate the expression of Ferritin (P<0.01),while decrease the level of LIP (P<0.001). The iron homeostasis in cells could be regulated by autophagy through affecting Ferritin expression and LIP level. Autophagy can alter sensitivity of ALL cells to ferroptosis activator Erastin, which suggestes that combining autophagy regulator with ferroptosis activator may be a new strategy for the treatment of chemotherapy-resistant ALL.

Loss of the redox mitochondrial protein mitoNEET leads to mitochondrial dysfunction in B-cell acute lymphoblastic leukemia

B-cell acute lymphoblastic leukemia (ALL) affects both pediatric and adult patients. Chemotherapy resistant tumor cells that contribute to minimal residual disease (MRD) underlie relapse and poor clinical outcomes in a sub-set of patients. Targeting mitochondrial oxidative phosphorylation (OXPHOS) in the treatment of refractory leukemic cells is a potential novel approach to sensitizing tumor cells to existing standard of care therapeutic agents. In the current study, we have expanded our previous investigation of the mitoNEET ligand NL-1 in the treatment of ALL to interrogate the functional role of the mitochondrial outer membrane protein mitoNEET in B-cell ALL. Knockout (KO) of mitoNEET (gene: CISD1) in REH leukemic cells led to changes in mitochondrial ultra-structure and function. REH cells have significantly reduced OXPHOS capacity in the KO cells coincident with reduction in electron flow and increased reactive oxygen species. In addition, we found a decrease in lipid content in KO cells, as compared to the vector control cells was observed. Lastly, the KO of mitoNEET was associated with decreased proliferation as compared to control cells when exposed to the standard of care agent cytarabine (Ara-C). Taken together, these observations suggest that mitoNEET is essential for optimal function of mitochondria in B-cell ALL and may represent a novel anti-leukemic drug target for treatment of minimal residual disease.

Britannin, a sesquiterpene lactone induces ROS-dependent apoptosis in NALM-6, REH, and JURKAT cell lines and produces a synergistic effect with vincristine.

Britannin, a Sesquiterpene Lactone isolated from Inula aucheriana, has recently gained attraction in the therapeutic fields due to its anti-tumor properties. This study was designed to evaluate the effect of this agent on Acute Lymphoblastic Leukemia (ALL) cell lines, either as a monotherapy or in combination with Vincristine (VCR). To determine the anti-leukemic effects of Britannin on ALL-derived cell lines and suggest a mechanism of action for the agent, we used MTT assay, Annexin-V/PI staining, ROS assay, and real-time PCR analysis. Moreover, by using a combination index (CI), we evaluated the synergistic effect of Britannin on Vincristine. We found that unlike normal Peripheral Blood Mononuclear Cells (PBMCs) and L929 cells, Britannin reduced the viability of NALM-6, REH, and JURKAT cells. Among tested cells, NALM-6 cells had the highest sensitivity to Britannin, and this agent was able to induce p21/p27-mediated G1 cell cycle arrest and Reactive Oxygen Specious (ROS)-mediated apoptotic cell death in this cell line. When NALM-6 cells were treated with Nacetyl-L-Cysteine (NAC), a scavenger of ROS, Britannin could induce neither apoptosis nor reduce the survival of the cells suggesting that the cytotoxic effect of Britannin is induced through ROS-dependent manner. Moreover, we found that a low dose of Britannin enhanced the effect of Vincristine in NALM-6 cells by inducing apoptotic cell death via altering the expression of apoptotic-related genes. Overall, our results proposed a mechanism for the cytotoxic effect of Britannin, either as a single agent or in combination with Vincristine, in NALM-6 cells.

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.

Prolonged Azacitidine Exposure Alters Immunophenotype in Myeloid Leukemia Cell Lines

Hypomethylating agents (HMA), namely azacitidine and decitabine, are used for treatment of MDS and AML. Although HMAs have demonstrated efficacy in a subset of patients, their mechanism of action is not well established. Some studies indicate that HMAs induce expression of specific target proteins, thus enhancing the efficacy of targeted therapies. For example, studies of HMAs used in combination with gemtuzumab ozogamicin (GO) suggest that they can apply additional epigenetic stress to AML cells and increase surface CD33 expression, thereby increasing the efficacy of GO. Despite suggestions of epigenetic modulation of target antigens, data on comprehensive response to HMAs is lacking. We proposed to evaluate the full extent of surface antigen expression in different AML cell lines after prolonged exposure to azacitidine.The dose and duration of azacitidine therapy used for prolonged exposure was established in Kasumi-1 cells (t(8;21), N822K KIT mutation). A daily dose of 250 nM allowed continued cellular proliferation for 18 days. We then treated two cell lines, Kasumi-1 and MV4-11 (t(4;11), FLT3 -ITD+) as above for 14-18 days. Cells were submitted for multi-dimensional flow cytometry (MDF) every 2-4 days.Prolonged azacitidine exposure resulted in broad changes in hematopoietic cell surface antigens, as demonstrated by MDF analysis. CD33, the premier cell surface target for AML immunotherapy, increased significantly in Kasumi-1 cells, approximately doubling by day 14 of treatment (p=0.003, Fig. 1). C -kit, or CD117, which is overexpressed in Kasumi-1 cells and is a potential target of tyrosine-kinase inhibitor (TKI) therapy in t(8;21) AML, was significantly decreased with azacitidine exposure (p <0.0001, Fig. 1). In contrast, treatment of MV4-11 cells resulted in significant decrease in CD33 with a corresponding increase in CD117 (Fig. 1).Kasumi-1 and MV4-11 both exhibited significant increases in surface CD38, CD13, and CD36, and significant decreases in CD64, CD123, and CD11b. Cell marker changes observed in only one cell line include significantly increased CD34 in Kasumi-1 and significantly increased CD56 in MV4-11. Many of the observed changes peaked after day 10 of treatment. No significant changes were seen in either line in CD7, CD14, CD16, CD19, CD45, and HLA-DR. Notably, the shared pattern of surface antigen changes in both Kasumi-1 and MV4-11 indicates that prolonged azacitidine exposure may induce a less mature myeloid phenotype, as typified by increased CD38 and decreased CD11b and CD123 (Fig. 1).The results above were validated in an experiment in which 4 leukemia cell lines were exposed to azacitidine as above for 10 days, with MDF analyses on days 5 and 10. Cell lines included Kasumi-1, MV4-11, K562 (t(9;22)), and the ALL cell line REH (t(12;21)). Similar trends in surface antigens for Kasumi-1 and MV4-11 were again observed. K562 cells exhibited increased CD38, CD36, CD7, CD14, and CD19, and decreased CD33, CD56, CD64, and CD45 (Fig. 1). No change was seen in K562 in CD117, CD13, CD11b, CD16, CD34, CD123, and HLA-DR (Fig. 1). Of these myeloid markers tested, REH cells had only a significant increase in CD13, a marker also seen in very early B-lymphocytes. Thus, HMAs do not appear to promote lymphoid to myeloid phenotype shift in REH.We observed broad changes in cell surface antigen expression in myeloid leukemia cell lines treated with prolonged azacitidine, which may signify changes in gene expression induced by hypomethylation. Among AML cell lines, prolonged azacitidine exposure resulted in a less mature immunophenotype. We observed significant changes in CD33 and CD117, both of which are considered potential therapeutic targets in AML. CD33 expression was increased by azacitidine exposure in Kasumi-1 cells, and reduced in MV4-11 and K562. C -kit was also noted to variably change in response to azacitidine. Downregulation of cell surface antigens is a known resistance mechanism to immunotherapeutic agents, including antibody-drug conjugates such as GO. Resistance to c -kit-targeted agents has been proposed to occur through a variety of mechanisms, including cell surface expression, therefore changes to c -kit levels induced by HMAs may be an important avenue of investigation in averting TKI resistance. The use of prolonged, low-dose hypomethylating agents warrants further investigation as a potential means to modulate surface protein levels for therapeutic benefit. [Display omitted] DisclosuresPardo:Hematologics Inc: Employment. Loken:Hematologics Inc: Employment, Equity Ownership. Brodersen:Hematologics Inc: Employment.

Obesity-Induced Chronic Inflammation Suppresses the Function of Non-Malignant Immune Cells While Upregulating PD-L1 on Human B-ALL Cell Lines

Obesity incidence continues to rise dramatically throughout the world. Currently, 20% of school-aged children (ages 6-19) and 35% of the adult population (> 20 years of age) in the United States are obese. Obesity-related complications contributes to various diseases including insulin resistance, type 2 diabetes, and cardiovascular diseases. In addition to these diseases, obesity is a risk factor for developing childhood leukemia and significantly increases mortality rates in pediatric patients. Obesity-associated chronic inflammation is postulated to contribute to the onset and propagation of leukemia; however, this hypothesis has remained largely unexplored in experimental settings. Therefore, we established an in vitro system to delineate how non-malignant immune cells and B-cell leukemia lines respond to cytokine stimulation. Specifically, dendritic cells (DC) were isolated from the spleens of C57BL/6 mice using anti-CD11c microbeads followed by MiniMACS purification and stimulated with IL-1β, IL-6, TNF-α, or LPS (as single agents or in combination) for 24 hours prior to assaying for the upregulation of CD80,CD86, and PD-L1 using flow cytometric analysis. We found that splenic DC stimulated with IL-1β or TNF-α upregulated both CD80 and CD86 in a dose-dependent manner. Surprisingly, both cytokines also induced PD-L1 expression on DC. We next tested the effects of cytokine stimulation on Nalm6, REH, and SEM human B-ALL cell lines. Only SEM cells expressed CD86, which was not modulated by cytokine stimulation. In contrast, stimulating REH and SEM cells with cytokines upregulated PD-L1 expression with TNF-α being the most potent inducer of the immune inhibitory receptor. In order to mimic a chronic inflammatory microenvironment in vitro, mouse DC cells were treated with individual pro-inflammatory cytokines for 24 hours prior to stimulation with LPS treatment for 24 hours before assaying for functional outcomes at 48 hours post-stimulation. We observed that LPS, IL-1β, and TNF-α treatments increased CD80 and CD86 expression. Surprisingly, LPS upregulation of both CD80 and CD86 was blocked on DC when cytokine treatment preceded LPS stimulation. Similarly, DC exposure to cytokines prior LPS treatment failed to induced CD40 and PD-L1 upregulation. To examine how adipocytes-produced inflammatory cytokines modulated these functional responses in non-malignant B cells, we isolated mouse B cells from spleens of C57BL/6 mice using anti-B220 microbeads followed by MiniMACS purification. Using a modified insulin Oleate (IO) media differentiation protocol, we were able to induce adipocyte differentiation from OP9 cells (a mouse bone marrow stromal cell line). Adipocytes differentiated in vitro morphologically phenocopied primary adipocytes, and they expressed a hallmark adipocyte marker Fatty Acid Binding Protein 4 (FABP4). Importantly, these cells produced copious amounts of TNF-α. When splenic B-cells were cultured with adipocyte-produced supernatants, we found that CD86 surface expression was downregulated while PD-L1 expression increased. In conclusion, we found that IL-1β and TNF-α potently induced CD80 and CD86 on splenic DC and B-cells in acute settings. Surprisingly, these cytokines also induced PD-L1 expression on these cells most likely to prevent the pathological effects of an uncontrolled immune response. In the chronic settings, we observed blunted innate immune responses to LPS stimulation. These observations suggest that chronic inflammatory microenvironments (such as those associated with obesity) will induce an immune suppressive state in splenic DC and B-cell populations. This hypothesis was supported by our observations that adipocyte-produced soluble factors also decreased CD86 expression and upregulated PD-L1 on splenic B-cells. Pro-inflammatory cytokines also potently induced PD-L1 expression on B-ALL cells. In all, our data suggest that chronic inflammation may promote leukemogenesis by both attenuating de novo immune responses and reducing the ability to effector T-cells to kill leukemia cells when engaged due to the upregulation of immune suppressive mechanisms. Disclosures No relevant conflicts of interest to declare.

CD9 Blockade Effectively Suppresses Disease Progression of Pediatric B-Precursor Acute Lymphoblastic Leukemia and Enhances Chemosensitivity

Despite major advances in risk-directed multi-agent chemotherapy, specific subtypes of high-risk and relapsed/refractory pediatric B-precursor acute lymphoblastic leukemia (B-ALL) remain associated with poor clinical outcomes, highlighting the need for development of novel and effective therapeutic strategies. CD9, a tetraspanin family protein, has been associated with metastasis and progression of various types of malignancies, but its prognostic relevance and therapeutic potential in pediatric B-ALL remain largely unknown. In a cohort of pediatric B-ALL cases, we identified that CD9+ patients had significantly lower 5-year overall (OS) and relapse-free survival (RFS) rates when compared with CD9- cases (OS: 81.5% vs 100%, P= 0.022; RFS: 70.7% vs 92.3%, P= 0.029). Subgroup analysis revealed remarkably poorer outcomes in CD9+ patients of the high-risk group (OS: 61.5% vs 100%, P= 0.042; RFS: 46.2% vs 88.9%, P= 0.045). A similar trend was also observed in patients of the intermediate-risk group but not in the standard-risk group. In univariate analysis, CD9 positivity, age <1 year, white cell count ≥100 × 10^9/L and poor prednisone response were associated with lower RFS rate. In multivariate analysis, CD9 positivity (HR = 6.0; P= 0.019) remained as an independent prognostic factor for lower RFS rate. The efficacy of targeting CD9 for treatment of pediatric B-ALL was evaluated in the NOD/SCID mouse xenograft model. Our results showed that administration of CD9 neutralizing antibody as a single agent substantially reduced multi-organ leukemic burden by >90% (P ≤ 0.037) and significantly prolonged survival (P ≤ 0.007) of animals xenografted with the intermediate-risk 697 (TCF3-PBX1+) and high-risk RS4;11 (MLL-AF4+), but not the standard-risk Reh (ETV6-RUNX1+) cell lines. Similarly, CD9 antibody treatment significantly decreased B-ALL progression in patient-derived xenografts with a wide spectrum of genetic and disease features, including high-risk infantile MLL-AF4+ cases as well as those with relapsed/refractory diseases (P ≤ 0.009). Importantly, CD9 antibody in combination with conventional chemotherapy consisting of vincristine, dexamethasone and L-asparaginase further prolonged animal survival, when compared to animals treated with CD9 antibody or chemotherapy alone (P ≤ 0.006). In vitro, CD9 antibody significantly reduced proliferation of B-ALL cells (P= 0.004) and markedly enhanced vincristine-, dexamethasone- and L-asparaginase-induced apoptosis (P ≤ 0.044). Mechanistic studies revealed that CD9 was involved in B-ALL migration and stromal adhesion by modulating the affinity of integrin very late antigen-4. Our results collectively suggest that expression of CD9 in pediatric B-ALL patients was associated with adverse survival outcomes and that CD9 blockade, in adjunct to chemotherapy, could be a promising strategy for treatment of high-risk and relapsed/refractory pediatric B-ALL, possibly mediated by disruption of leukemia-stroma interaction in the bone marrow microenvironment. DisclosuresNo relevant conflicts of interest to declare.

Genomic and Epigenetic Effects of DNA Methyltransferase Inhibition in Acute Lymphoblastic Leukemia

Background: Children with acute lymphoblastic leukemia (ALL) who relapse are more resistant to chemotherapy. We have previously identified a unique gene expression signature associated with relapsed leukemic blasts and showed that relapse samples have higher level of CpG methylation compared to diagnosis. We also demonstrated that treatment with DNMT inhibitor, decitabine, alone or in combination with the HDAC inhibitor, vorinostat restored blast chemosensitivity in vitro . These observations led to the hypothesis that global methylation status is a key regulator of drug resistance.Method: To discover the pathways affected by changes in global methylation and chromosomal architecture, we performed ChIPseq for the CCCTC-binding factor (CTCF) and the histone marks H3K9ac, H3K27ac and H3K27me3 as well as RNAseq, and DNA methylation in the B-ALL cell line Reh treated with 1µM decitabine versus DMSO for 3 days in triplicates. ChIPseq data was analyzed using MACS2 broad setting for peak calling, GENCODE for gene annotations and diffbind for differences between the two groups. Promoter regions were inferred to be +/-3KB from TSS and the ROSE algorithm was used on the H3K27ac mark to define enhancers and superenhancers. Analysis of differential gene expression was performed using DESeq2 Bioconductor package and the 850k methylation data was analyzed using ‘Minfi’ Bioconductor package. For all data sets, fold changes of 1.5 or more and p<0.05 (FDR of 5% for the ChIPseq data) were considered significant.Results: As expected, decitabine treatment resulted in a global loss of CpG methylation and there were 1286 genes upregulated and 1003 genes downregulated by 1.5 fold or more in treated versus untreated cells. Interestingly, there was a subset of CpG sites which had a significant gain of methylation, and gene ontology biological process analysis of those sites using DAVID Bioinformatics identified, among others, genes involved in mitotic cell cycle and MAPK activation. ChIPseq data analysis show that more than 90% of the peaks remain unchanged for the activation mark H3K27ac and the repressive mark H3K27me3 between decitabine-versus DMSO-treated Reh cells. However decitabine treatment resulted in a slight variation in the genomic distribution of the overall peaks. H3K27ac mark occupied 28% of the promoter region after decitabine treatment as compared to 23% in untreated state and likewise H3K27me3 occupancy changed from 11% to 8% at the promoter region with treatment with decitabine. The most impacted histone mark was H3K9ac where 40% of the global peaks called in the untreated cells were lost upon DNMTi treatment. However most of the losses of the H3K9ac mark were located outside of promoters resulting in 56% of these activation marks in promoter regions in treated cells compared to 35% in the untreated cells. Unlike the expected gain of CTCF from lower DNA methylation, there was a 30% reduction of CTCF binding sites with decitabine treatment. CTCF was disproportionately lost at promoter regions compared to gene body and intergenic regions. Integrative analysis of changes in gene expression, CpG methylation, histone marks reveal that up to 60% of their gene expression changes can be directly attributed to the observed epigenetic changes with the greatest impact from changes in DNA methylation and H3K9ac. The gene expression data was also compared to our previously published relapsed gene expression signature on a large cohort of diagnosis-relapse primary samples. We found that decitabine reversed the expression of 74 genes which are typically downregulated in blasts at relapse and 57 genes which are typically upregulated in relapsed blasts. Decitabine treatment led to two predominant epigenetic modifications: loss of CpG methylation with corresponding de-repression of genes typically downregulated in relapse samples (primarily proapototic and antiproliferative genes) and loss of H3K9ac at promoters of antiapoptotic genes.Conclusions: We show that DNMTi not only impacts CpG methylation but also histone status, likely chromosomal architecture, and gene expression in B-ALL cells and can reverse expression of a subset of the relapse gene expression signature. Further study and validation will lead to a better description of epigenetic events in relapsed ALL that may guide future therapy. DisclosuresNo relevant conflicts of interest to declare.

The Role of MMSET E1099K Mutation in Gene Dysregulation, Drug Resistance and Clonogenicity in Acute B-Lymphoblastic Leukemia

Background: While outcomes for pediatric patients with newly-diagnosed acute B-lymphoblastic leukemia (ALL) have dramatically improved over the past 20 years, relapse remains a major cause of treatment failure and death. Discovering the underlying biological pathways that lead to drug resistance and relapse is therefore a top priority. MMSET (NSD2 / WHSC1) encodes a histone methyltransferase that dimethylates histone 3 lysine 36 (H3K36me2) with subsequent decrease in histone 3 lysine 27 trimethylation (H3K27me3). Overexpression of MMSET, as in the t(4;14) translocation in multiple myeloma (MM), has been shown to affect proliferation, gene expression, chromatin accessibility and DNA damage repair. Mutations in the SET domain of MMSET resulting in a glutamate to lysine substitution at amino acid 1099 (p.E1099K) have been recognized as one of the most common mutations in relapsed ALL in children. While it has been shown that this leads to increased enzyme activity, the downstream effects of this mutation and the underlying mechanisms of tumor escape are poorly understood. Understanding the biological pathways affected by this mutation is key to discovering new therapies to improve outcomes.Objective: To determine the role of the MMSET p.E1099K mutation in cell proliferation, drug resistance and gene regulation.Design/Method: B-lineage REH and 697 ALL cell lines were transduced with lentiviral vectors overexpressing either wild-type (WT) or mutant (p.E1099K) MMSET or an empty vector (EV) control. Western Blot analysis confirmed overexpression of MMSET as well as increase in H3K36me2 and decrease in H3K27me3 in overexpressed WT and mutant cell lines. The generated cell lines were then plated with and without stroma and evaluated for differences in apoptosis and proliferation in response to chemotherapeutic agents commonly used in the treatment of ALL (e.g. mercaptopurine, thioguanine, methotrexate, prednisone, cytarabine, etoposide, doxorubicin and L-asparaginase). Clonogenic growth in vitro was also assessed using MethoCultTM media. Differences in gene regulation in the REH cell lines were assessed by performing ChIPseq for CCCTC-binding factor (CTCF) and histone marks (H3K9Ac, H3K27Ac, H3K36me2 and H3K27me3) and the results were correlated with RNAseq data.Results: The transduced WT, mutant and EV cell lines did not display differences in proliferation, cell cycle regulation or adhesion. Moreover we were unable to detect any intrinsic drug resistance between the cell lines with respect to apoptosis or proliferation both with and without stroma in vitro . There was, however, a marked increase in clonogenicity in the cell lines overexpressing WT or mutant MMSET compared to the EV control (348% and 285% increased colony number, respectively) when plated in MethoCultTM media.Significant differences in the epigenetic landscape were observed between WT and mutant vs. EV control. As previously described in t(4;14) MM cell lines, over 99% of peaks for the H3K27me3 and H3K36me2 were lost in both the overexpressed WT and mutant cell lines compared to the EV control. However, whereas the decrease in peaks in H3K27me3 represents an overall loss of the mark, the lower peak number for H3K36me2 is actually due to widespread enrichment of the mark throughout the genome.Moreover, RNAseq data revealed significant differences in gene expression between the three cell lines. Notably, the overexpressed WT and mutant cell lines revealed 83 and 90 upregulated genes and 44 and 99 downregulated genes, respectively, compared to EV control. Notably, 70% of upregulated genes and 80% of downregulated genes were shared between cell lines. When compared with previously published data on t(4;14) MM cell lines, the genes upregulated by overexpressed WT or mutant MMSET appear to be unique to leukemia. Interestingly, ETV5, which is involved in transcriptional regulation , is upregulated in both our cell lines and MM cell lines.Conclusion: Catalytically active MMSET rendered by the p.E1099K substitution in ALL leads to an altered chromatin landscape and distinct gene expression signature (distinct from the t(4;14)) but does not result in intrinsic drug resistance in vitro . The increased clonogenic potency may suggest an increased leukemic stem cell population but understanding the precise mechanism for clonal expansion and relapse is the subject of additional in vitro and in vivo experiments. DisclosuresNo relevant conflicts of interest to declare.

Knockdown of GPSM1 Inhibits the Proliferation and Promotes the Apoptosis of B-Cell Acute Lymphoblastic Leukemia Cells by Suppressing the ADCY6-RAPGEF3-JNK Signaling Pathway

B-cell acute lymphoblastic leukemia (B-ALL) is the common type of blood cancer. Although the remission rate has increased, the current treatment options for B-ALL are usually related to adverse reactions and recurrence, so it is necessary to find other treatment options. G protein signaling modulator 1 (GPSM1) is one of several factors that affect the basic activity of the G protein signaling system, but its role in B-ALL has not yet been clarified. In this study, we analyzed the expression of GPSM1 in the Oncomine database and found that the GPSM1 levels were higher in B-ALL cells than in peripheral blood mononuclear cells (PBMCs). Analyses of the Gene Expression Profiling Interactive Analysis (GEPIA) demonstrated that patients with high GPSM1 levels had shorter survival times than those with low levels. Additionally, gene set enrichment analysis (GSEA) suggested that GPSM1 was positively correlated with proliferation, G protein-coupled receptor (GPCR) ligand binding, Gαs signaling and calcium signaling pathways. In further experiments, GPSM1 was found to be highly expressed in Acute lymphoblastic leukemia (ALL) cell lines, and downregulation of GPSM1 inhibited proliferation and promoted cell cycle arrest and apoptosis in BALL-1 and Reh cells. Moreover, knockdown of GPSM1 suppressed ADCY6 and RAPGEF3 expression in BALL-1 and Reh cells. Furthermore, we reported that GPSM1 regulated JNK expression via ADCY6-RAPGEF3. The present study demonstrates that GPSM1 promotes tumor growth in BALL-1 and Reh cells by modulating ADCY6-RAPGEF3-JNK signaling.

Synthesis, molecular modeling, and biological evaluation of novel imatinib derivatives as anticancer agents.

Different derivatives of imatinib were synthesized by a 3-step reaction method. The structures of the new compounds were characterized by spectroscopic methods. For quantitative evaluation of the biological activity of the compounds, MTT assays were performed, where four BCR-ABL negative leukemic cell lines (Jurkat, Reh, Nalm-6 and Molt-4), one BCR-ABL positive cell line (K562), and one non-leukemic cell line (Hek293T) were incubated with various concentrations of the derivatives. Although imatinib was specifically designed for the BCR-ABL protein, our results showed that it was also effective on BCR-ABL negative cell lines except for Reh cell line. Compound 9 showed lowest IC50 values against Nalm-6 cells as 1.639 μM, also the values of Compound 10 for each cell were very close to imatinib. Molecular docking simulations suggest that except for compound 6, the compounds prefer a DFG-out conformation of the ABL kinase domain. Among them, compound 10 has the highest affinity for ABL kinase domain that is close to the affinity of imatinib. The common rings between compound 10 and imatinib adopt exactly the same conformation and same type of interactions in the ATP binding site with the ABL kinase domain.