Dynamin-2 (DNM2) is a GTPase essential for intracellular vesicle formation and trafficking, cytokinesis and receptor endocytosis. Mutations in DNM2 are common in early T-cell precursor acute lymphoblastic leukemia. However, DNM2 expression in other types of ALL are not reported. We studied DNM2 mRNA level in adults with B- and T-cell ALL. We found DNM2 is more highly expressed compared with normals in both forms of ALL. High DNM2 expression is associated with some clinical and laboratory features, inferior outcomes and with leukaemia cell proliferation. We also found Ikaros directly binds the DNM2 promoter and suppresses DNM2 expression. Consequently IKZF1 deletion is associated with high DNM2 expression. Conversely, casein kinase-2 (CK2)-inhibitor increases Ikaros function thereby inhibiting DNM2 expression. Inhibiting DNM2 suppresses proliferation of leukemia cells and synergizes with CK2 inhibition. Our data indicate high DNM2 expression is associated with Ikaros dysregulation and may be important in the development of B-ALL.
Dynamin-2 (DNM2) is a microtubule-associated GTPase essential for intracellular vesicle formation and trafficking, cytokinesis, and receptor endocytosis. Recent reports suggest over-expression of DNM2 promotes cancer cell growth, migration and invasion in diverse cancers1,2,3. One DNM2 mutant, DNM2V265G, is associated with cancer development in mice4. Recently, mutations in DNM2 were detected in early T-cell precursor acute lymphoblastic leukemia (ALL)5,6. There are no studies of DNM2 mRNA levels in ALL.
IZKFI encodes the DNA-binding zinc finger protein Ikaros essential for normal hematopoiesis and immune development7,8,9,10,11. Ikaros is also a tumor suppressor gene in acute B- and T-cell ALL11,12,13,14. Recently, we reported the Ikaros global binding profiling in ALL cells. We found Ikaros regulates expression of its targets through chromatin remodeling in ALL15,16,17,18. We also found CK2-inhibitors increase tumor suppressor activity of Ikaros and act as a functional activator of Ikaros15,16,17. Our ChIP-seq data indicate Ikaros binding peaks in the promoter region of DNM2. However, it is unclear how Ikaros regulates DNM2 expression. We studied correlations between DNM2 mRNA level and outcomes in adults with B- and T-cell ALL. Our data suggest high expression of DNM2 with consequent Ikaros dysfunction is associated with development of B-cell ALL.
Clinical and laboratory variables in subjects with high and low DNM2 expression
DNM2 mRNA levels in bone marrow samples from adults with ALL, especially those with B-cell ALL, were significantly higher than those in normals (Fig. 1A). We compared clinical and laboratory variables in subjects with B- and T-cell ALL divided into cohorts with high or low DNM2 mRNA levels (Tables 1 and 2). In B-cell ALL, high DNM2 mRNA levels were associated with a WBC ≥ 30 × 10E + 9/L compared to low DNM2 expression (79% vs. 42%; P = 0.003). This associated was confirmed in multivariate analyses (HR 4.56, 95% confidence interval [CI] 1.09, 19.01; P = 0.037; Table 1). The high DNM2 expression cohort also had a higher frequency of lymph-adenopathy compared with the low DNM2 expression cohort (61% vs. 23%; P = 0.002) confirmed in multivariate analyses (HR 7.245, [1.74, 30.13]; P = 0.006; Table 1). There were no significant associations between clinical and laboratory variables in the high versus low DNM2 expression cohorts in subjects with T-cell ALL (Table 2).
Correlation between DNM2 expression and clinical outcomes
Subjects with B-cell ALL and high DNM2 expression had briefer median RFS than those with low DNM2 expression (9 months [3.7, 14.3 months] vs. 14 months [0, 37.9 months]; P = 0.095; Fig. 1B). 5 year survival was significantly briefer in those with high DNM2 expression (13 months [7.8, 18.2 months] vs. 33 months [20.4, 45.6 months]; P = 0.017; Fig. 1C). There was no significant difference in median EFS between the cohorts (9 months [5.1, 12.9 months] vs. 11 months [7.4, 14.6 months]; P = 0.319; Fig. 1D). There were no significant associations between DNM2 expression and any outcome in subjects with T-ALL (Supplemental Figure 2 and Table 2).
Ikaros binds to the DNM2 promoter and regulates its expression in ALL
To address the potential mechanism underlying high DNM2 expression we analyzed transcription factor motifs in the DNM2 promoter region. ChIP-seq data identified Ikaros binding peaks in the DNM2 promoter region in Nalm6 B-ALL (Fig. 2A) and primary B-cell ALL cells (Supplemental Figure 3)15,16. Ikaros binding was confirmed by qChIP assay (Fig. 2B and C). Weak binding was also found in U-937 AML cells and Molt-4 T-cell ALL cells (Fig. 2B). Ikaros suppressed promoter activity of DNM2 by luciferase reporter assay (Fig. 3A). These data indicate a direct effect of Ikaros on DNM2 transcription. Expression of Ikaros suppresses DNM2 mRNA levels in Nalm6 (Fig. 3B) and CEM cells (Fig. 3C). Conversely, efficient Ikaros knockdown increased DNM2 expression in Nalm6 (Fig. 3D) and CEM cells (Fig. 3E). Treating Nalm6 and CEM cells with TBB suppressed DNM2 mRNA levels in a dose-dependent manner detected by qPCR (Fig. 4A) and protein levels by western blotting (Fig. 4B). CK2 knockdown with shRNA also induced suppression of DNM2 expression in Nalm6 (Fig. 4C) and CEM cells (Fig. 4D). Ikaros knockdown with shRNA blocked the TBB-induced decrease of DNM2 expression in Nalm6 and CEM (Fig. 4E and F) cells. These data indicate DNM2 is the direct target of Ikaros and that Ikaros suppresses DNM2 expression in B- and T-cell ALL.
Correlation of IKZF1 deletion with DNM2 high expression in ALL cells
We next analyzed a possible correlation between IKZF1 levels with DNM2 in primary ALL cells19,20,21. We found IKZF1 mRNA levels were inversely-correlated with high DNM2 expression in both cohorts (Supplemental Figures 4 and 5). We also found DNM2 expression was significantly higher in B-cell ALL cells with Ik6 (13.58 ± 5.53 vs. 3.28 ± 0.92; P = 0.0048; Fig. 5A). Frequency of Ik6 in subjects with high DNM2 expression was significantly higher than in subjects with low DNM2 expression (54% vs. 21%; P = 0.004) confirmed in multivariate analyses (HR 6.15 [1.40, 27.00]; P = 0.016; Table 1). These data suggest IKZF1 deletion may contribute to high DNM2 expression. Furthermore, treatment of primary B- and T-cell ALL cells with TBB increased Ikaros binding to the DNM2 promoter region compared with normals (Fig. 5B). qPCR assays showed TBB treatment inhibited expression of DNM2 mRNA in a dose-dependent manner (Fig. 5C). These results indicate Ikaros binds to the DNM2 promoter and treatment with TBB, which enhances Ikaros tumor suppressor activity, suppresses DNM2 expression.
Enhanced Ikaros activity from CK2-inhibition increases H3K9me3 at the DNM2 promoter
We reported Ikaros regulates gene expression through chromatin remodeling16. To further explore the epigenetic mechanism by which Ikaros regulates DNM2 expression we performed ChIP assay and amplified the resulting DNM2 promoter sequences. Our data indicate TBB treatment significantly increases binding of Ikaros to the DNM2 promoter region compared with untreated Nalm6 B-cell ALL cells and with CEM T-cell ALL cells (Fig. 6A). TBB treatment also enhanced binding of H3K9me3 at the DNM2 promoter region in Nalm6 and CEM cells (Fig. 6B). Results were similar with primary B- and T-cell ALL cells (Fig. 6C). There was no enrichment of other histone modification markers in the DNM2 promoter region (data not shown).
MiTMAB suppresses cell proliferation and synergizes with CK2-inhibitors in ALL
We used the WST-1 cell proliferation assay to test whether targeting DNM2, with MiTMAB, a DNM2-inhibitor, had an anti-leukemia effect in ALL cells22. Treatment with MiTMAB had a dose-dependent effect on proliferation of Nalm6 B-cell ALL cells (Fig. 7A) and CEM T-cell ALL cells (Fig. 7B). In contrast, treatment with Dynasore, a GTPase-inhibitor which suppresses dynamin activity and prevents endocytosis, had no significant effect on cell proliferation (data not shown)23,24. Because CK2-inhibition results in reduced expression of DNM2 we tested whether the CK2-inhibitors TBB and CX-4945 with MiTMAB altered cell proliferation. CX-4945 (4 μM) and various doses of MiTMAB resulted in less proliferation of Nalm-6 (Fig. 7A) and CEM cells (Fig. 7B) compared to MiTMAB alone (Fig. 7A green line vs. red line). Using the CalcuSyn assay we found treatment with CX-4945 and MiTMAB were synergistic in inhibiting proliferation of Nalm-6 (Fig. 7C) and CEM (Fig. 7D) cells25. Similar result was observed for TBB (data not shown). These data indicate targeting DNM2 transcription significantly suppresses proliferation of ALL cells and that inhibiting DNM2 with TBB, a CK2–inhibitor restores Ikaros function.
High DNM2 expression was seen in adults with B- and T-cell ALL. In persons with B-cell ALL, high DNM2 expression is associated with cell proliferation, extra-medullary leukaemia, an increased relapse risk and briefer survival. We also found DNM2 expression is significantly higher in subjects with IKZF1 deletion. Ikaros directly suppresses DNM2 expression in ALL cells and a CK2-inhibitor which restores Ikaros function and suppresses DNM2 expression in an Ikaros-dependent manner in leukemia cells by recruiting the repressive histone marker-H3K9me3. Importantly, we observed DNM2-inhibitors suppresses proliferation of leukemia cells and is synergistic with CK2-inhibitors. Our findings suggest an oncogenic role for high DNM2 expression in ALL, a possible therapeutic role for DNM2–inhibitors alone or combined with CK2 inhibitors in B-cell ALL (Fig. 8).
Others have reported DNM2 mutations in adults with ALL but DNM2 expression was not studied5,6,26. Although we did not detect DNM2 mutations in adults with B-cell ALL (data not shown) we found significantly higher expression of DNM2. In contrast, we detected DNM2 mutations in 4 adults with T-cell ALL27. High DNM2 expression is also seen in subjects with T-cell ALL, but DNM2 expression in subjects with mutations is difficult to evaluate because of few subjects. Although we found clinical correlations between DNM2 expression and laboratory variables in B-cell ALL we did not find this in T-cell ALL, perhaps because the few subjects studied gave us little power to detect correlations.
High DNM2 expression is reported in human cancers and is associated with cancer progression and metastasis28,29, and cancer cell proliferation30. Oncogenic mechanisms underlying high DNM2 expression are partially understood. For example, DNM2 enzymatic activity and proper intra-cellular localization are required for extracellular matrix degradation by invasive cancer cells31. DNM2 is required for the endocytosis of several proteins associated with cancer motility and invasiveness32,33 and for endocytosis of several oncogenic receptors34,35. Furthermore, DNM2 regulates Golgi structure and vesiculation during the secretory process which can affect trafficking of other carcinogenic signaling molecules36. Finally, DNM2 interacts with F-actin and actin dynamics, actin-associated proteins, and molecules that induce or sense membrane curvature as well as expression of angiogenic receptors37,38. Our study shows high DNM2-expression in persons with ALL. We and others think DNM2 may be important to regulate membrane trafficking of oncogenes and/or oncogenic receptors signaling. It is reported that DNM2 is critical for endocytosis and/or internalization of IL7R, T cell receptor (TCR) and Notch1 ligand Delta-like1 (Dll1), which will trigger signaling activation in the receptor-presenting cells and could be important in the development of ALL4,7,38,39,40,41. Moreover, DNM2 is involved in control of cytokinesis and acts like a signal transducing GTPase affecting transcriptional regulation42. We observed the MiTMAB suppresses the cell proliferation of ALL cells, which is consistent with dynamin inhibitor (MiTMAB)-induced apoptosis in human Jurkat T cells43. We also observed that DNM2 is localized predominately in the cytoplasm with a weak nuclear localization in ALL cells (data not shown). These data suggest a possible nuclear mechanism underlying the oncogenic effect of DNM2 high expression in ALL although it needs to be further clarified.
It is largely unknown how DMN2 expression is regulated44. Our current study suggests that Ikaros suppresses transcription of DNM2 in leukemia cells. We observed Ikaros directly binds to and suppress DNM2 expression. Although Ikaros is dysfunctional in ALL, CK2-inhibitor suppresses DNM2 expression by restoring Ikaros function by chromatin re-modeling. These findings may explain the upstream mechanism underlying high DNM2 expression. We also observed a synergistic effect of a DNM2-inhibitor with a CK2-inhibitor in suppressing proliferation of B-ALL cells. These data suggest a possible role for inhibiting DNM2 as a therapy of adult ALL.
Materials and Methods
Subjects and samples
Between June 2008 and June 2015, 123 consecutive subjects with newly-diagnosed ALL (age 14–77 years old) were studied at the First Affiliated Hospital of Nanjing Medical University (72 had B-cell and 51, T-cell ALL). Diagnoses were based on the WHO Diagnosis and Classification of ALL (2008). The written informed consent was provided by all subjects. The study was approved by the Ethics Committee of the First Affiliated Hospital of Nanjing Medical University, Jiangsu Province Hospital, Nanjing, China, with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. There were no studies with animals.
Therapy details are published protocol (CALLG2008)45. Induction was with VDCLP (vincristine, daunorubicin, cyclophosphamide, L-asparaginase, prednisone). Early consolidation used CAT (cyclophosphamide, cytarabine, thioguanine), high-dose methotrexate/L-asparaginase, and mitoxantrone. Late consolidation used VDLP (vincristine, daunorubicin, L-asparaginase, prednisone), COATD (cyclophosphamide, vincristine, cytarabine, epipodophyllotoxin and dexamethasone), high-dose methotrexate/L-asparaginase, epipodophyllotoxin andcytarabine. Maintenance therapy used 6-mercaptopurine and methotrexate. Subjects with BCR/ABL1-positive ALL received Imatinib from day 15 of introduction therapy.
Cytogenetic and molecular analyses
Cytogenetics and detection of the most common Ikaros deletion, Ikaros 6 (Ik6) were analyzed as described17. qPCR was performed on StepOne Plus Real-time PCR system (Applied Biosystem-Thermofisher, Foster, CA, USA). Gene expression values of genes of interest (GOI) were achieved in each sample by a formula derived from a scatter graph of Ct values from serial dilutions of a template standard as described17,28. Expression levels of GOIs were normalized to housekeeping genes expressed as gene expression value of GOI/18 s rRNA. Subjects were allocated in a high or low DNM2 expression cohort (4th quartile vs. 1st–3rd quartiles) with a cut-off value (1.619) was determined by SPSS 17.017.
qPCR for DNM2 expression was analyzed as above in Nalm6, CEM cells and primary ALL cells. Results were normalized to those obtained with 18 s rRNA and presented as fold-induction over vector controls. Primers: 18s rRNA, Sense:5′-GTAACCCGTTGAACCCCATT-3′, Anti-sense: 5′-CCATCCAATCGGTAGTAGCG-3′; DNM2 Sense: 5′-TCAGGACCGGGCTTTTCA-3′, Anti-sense: 5′-CGACCTGCTTTTTCACAATGG-3′.
Cell culture, plasmids and retroviral gene transfer
The Nalm6 cell line is previously described46. The CCRF-CEM (CEM), MOLT-4 and U-937 cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA). Cells were cultured in RPMI-1640 medium (Cellgro, Tewksbury, MA, USA) supplemented with 10% fetal bovine serum (Hyclone, Logon, Utah, USA). HEK 293T cells were cultured in DMEM (Cellgro) supplemented with 10% fetal calf serum and 1% L-glutamine (Cellgro). Cells were incubated at 37 °C in a humidified atmosphere with 5% CO2. Primary human B- and T-cell ALL cells were cultured in RPMI–1640 medium (Cellgro) supplemented with 10% fetal bovine serum (GE-Hyclone, Logon, Utah, USA). 4,5,6,7-Tetrabromobenzotriazole (TBB) and CX-4945 were purchased from Sigma (St. Louis, MO, USA). Cells were cultured with or without TBB and collected for total RNA isolation. Human IKZF1 retroviral construct and retroviral production was described15,17,47,48.
Promoter of DNM2 (−1000bp to +200 bp) was cloned into pGL4.15 vector (Promega, Madison, WI, USA). The transient luciferase assay was performed in HEK293T cells using the Promega luciferase assay reagents and measured with a luminometer according to the manufacturer instructions15,17. The firefly luciferase activities were calculated as fold-change relative to values obtained from pGL4.15 vector only control cells, and expressed as a percent of pcDNA3.1-Ikaros transfection-induced luciferase activity vs. the pcDNA3.1 vector. All transfection and reporter assays were performed independently in triplicate at least three times.
Quantitative Chromatin Immune precipitation (qChIP)
qChIP assays were performed by incubating chromatin with antibodies against Ikaros15,16,17, H3K9me3 (Abcam, Cambridge, MA, USA) or normal rabbit IgG (Abcam) as a control15,16,17,48. Enrichment of the ChIP sample over input was evaluated by qPCR with ≥ 3 replicates using specific primers in the promoter region of DNM2(forward: 5′-ACCGCGGGATGGAAGAG-3′, reverse: 5′-TGAAGGCGTCCTGCAGTTT-3′). Relative concentration of the qPCR product is presented as the fold change of the level of DNA-Ikaros and DNA-H3K9me3 samples compared with controls.
IKZF1 shRNA knockdown
Nalm6 and CEM cells were transiently transfected with human IKZF1 shRNA constructs in the GFP vector (pGFP-v-RS) (OriGene) using the Neon Transfection System (Invitrogen, Carlsbad, CA, USA). We used scrambled 29-mer shRNA cassette in the pGFP-v-RS vector as a control. Knockdown of Ikaros was confirmed by IKZF1 mRNA level15,17,31. Primers used for qPCR are 5′-GGCGCGGTGCTCCTCCT-3′ (IKZF1-F) and 5′-TCCGACACGCCCTACGACA-3′(IKZF1-R).
Cell lysates were prepared as described15,47,48. 20 μg protein was boiled in SDS sample buffer for 10 min., samples run on SDS-PAGE and transferred to the membrane which were blocked with 5% non-fat dry milk at 24 °C for 1 h and incubated overnight at 4 °C with primary antibody (anti-DNM2, 1:1000; Abcam) or anti-actin (1;1000, Santa Cruz, Dallas, TX, USA). The membrane was washed and incubated with goat anti-rabbit IgG conjugated to horseradish peroxidase (1:3000) at 24 °C for 2 h. Blots were developed by the enhanced chemi-luminescence (ECLPlus, Amersham, Arlington Heights, IL, USA) according to the manufacturer’s instructions.
Cell Proliferation Assay
Cell proliferation was assayed as described15,48. Briefly, the colorimetric assay (WST-1 reagent) from Roche Life Science (Indianapolis, IN, USA) was performed in 96-well white clear bottom plates (Costar, 3603). 10E + 4 cells were seeded per well with no or PBIT treatment at the indicated concentration and cultured for 72 h. WST-1 reagent was added (10 μl/well) for 4 h and absorbance at 440 nm was measured using a Synergy H1 Hybrid Reader (BioTek. Winooski, VT, USA).
Median differences between the cohorts were evaluated using a Mann–Whitney U-test. Frequency differences were analyzed using uni- and multivariate Cox model. Relapse-free survival (RFS), event-free survival (EFS) and overall survival (OS) were estimated by the Kaplan-Meier method and compared by log-rank test. The Starting point for the observation time for EFS and OS were date of diagnosis. Death in induction, resistance, relapse, death in continuous complete remission or new cancer were considered events in EFS calculation. RFS was estimated for subjects achieving complete remission (CR) starting at the time remission was declared. Living subjects were censored for survival at last follow-up. Statistical analyses used SPSS version 17.0. Data were represented as mean value with bars representing the standard error of the mean (SEM). Determinations of statistical significance were performed using a Student t-test for comparisons of two groups or using analysis of variance (ANOVA) for comparing multiple groups.
How to cite this article: Ge, Z. et al. Targeting High Dynamin-2 (DNM2) Expression by Restoring Ikaros Function in Acute Lymphoblastic Leukemia. Sci. Rep. 6, 38004; doi: 10.1038/srep38004 (2016).
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Supported, in part, by The National Natural Science Foundation of China (81270613, 30973376), Jiangsu Province Key Medical Talents (RC2011077), Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry (39th), China Postdoctoral Science Foundation (20090461134), special grade of the financial support from China Postdoctoral Science Foundation (201003598), Six Great Talent Peak Plan of Jiangsu (2010-WS-024), Scientific Research Foundation for the Returned Overseas Chinese Scholars, Nanjing Municipal Bureau of Personnel (2009), Southeast University Basic Research Fund (2242016k40143; to Z.G.). Also supported, in part, by Four Diamond Foundation of Pennsylvania State University, PA, USA (to S.D. and C.S.). R.P.G. acknowledges support from the National Institute of Health Research (NIHR) Biomedical Research Centre funding scheme.
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