High Levels Of FLT3 Ligand (FL) Reverse Etoposide Resistance In FLT3-Mutant Acute Leukemia Via Substrate Inhibition: Implications For Treatment

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1284-1284
Author(s):  
Yarden S Fraiman ◽  
Colleen E. Annesley ◽  
Daniel Magoon ◽  
Di Sun ◽  
Patrick Brown
Keyword(s):  
Cell Cycle ◽  
Acute Leukemia ◽  
Cell Lines ◽  
Plasma Levels ◽  
Pediatric Patients ◽  
Conflicts Of Interest ◽  
Peak Plasma ◽  
Substrate Inhibition ◽  
Leukemia Cells ◽  
Acute Leukemias

Abstract Background FLT3 is expressed in most human acute leukemias. When activated by FL, wild type (wt) FLT3 dimerizes and initiates downstream signals that result in proliferation and inhibition of apoptosis and differentiation. Activating FLT3 mutations (internal tandem duplications (ITDs) or point mutations) are common in AML and rare in ALL. ITD mutations confer a poor outcome in AML. In vitro, mutant FLT3 signaling can be further enhanced by binding of FL. Peripheral blood (PB) plasma FL levels rise in adults with AML, peaking about two weeks after initiation of chemotherapy. We sought to determine plasma levels of FL in pediatric patients after chemotherapy, and the functional effect of various levels of FL on both wt and mutant FLT3 leukemia cells. Methods FL levels were measured using FL ELISA on plasma samples (n=352) isolated from PB of children (n=75) enrolled on 4 multi-center acute leukemia clinical trials. Functional studies were performed on AML and ALL cell lines with wt FLT3 (HL60, RS4;11, SEMK2, and KOPN-8) and mutant FLT3 (MOLM14, MV4-11, and HB-1119). 72 hr etoposide IC50 was determined by WST-1 for each line. Cells were plated (250,000 cell/mL) for 72hr at etoposide IC50 in RPMI 1640 along with increasing concentrations of recombinant human FL (62.5 to 4,000 pg/ml). Cell cycle and apoptosis were analyzed using propidium iodide staining and annexin V/7-AAD binding, respectively. To explore the mechanism of FL effects, Ba/F3-ITD cells were incubated for 72hr in serum-free conditions with either 4,000 pg/mL (“high”), 62.5 pg/mL (“low”), or no FL. After washing, total and phosphorylated FLT3 protein levels were determined by Western blot. Results Pediatric patients receiving chemotherapy for the treatment of acute leukemia demonstrate a pattern of plasma FL rise with low levels at baseline (mean 41 pg/ml) and peak levels at day 11-14 following initiation of therapy (mean: 1,190 pg/mL; max: 5,783 pg/mL)(Fig 1A). Cell lines with FLT3 activating mutations selectively demonstrate resistance to etoposide-induced apoptosis (Fig 1B) and G2/M cell cycle arrest (Fig 1C) at low concentrations of FL (62.5 pg/mL). Dose-dependent reduction of etoposide resistance is seen with increasing concentrations of FL up to 4,000 pg/mL, suggesting that optimal etoposide-induced killing of FLT3-mutant leukemias may occur when FL plasma levels are at their peak. Ba/F3-ITD cells pre-incubated with peak concentrations of FL showed diminished baseline FLT3 phosphorylation, suggesting that the interaction of FL and FLT3/ITD exhibits substrate inhibition kinetics and results in a loss of FLT3/ITD-induced activation with high level FL exposure, thus providing a mechanistic basis for the observed loss of etoposide resistance. Conclusions Plasma FL rises to peak levels 11-14 days after initiation of chemotherapy. Through substrate inhibition of mutant FLT3 enzymatic activity, peak FL levels may reduce the etoposide resistance that characterizes FLT3-mutant leukemia cells exposed to pre-chemotherapy levels of FL. Thus, introduction of etoposide in a “time sequential” manner during periods of peak plasma FL levels may enhance killing of residual chemoresistant FLT3-mutant leukemia cells. Disclosures: No relevant conflicts of interest to declare.

Elevated FoxM1 Expression Promotes Cell Cycle Progression by Induction of KIS Expression and Contributes to the Development of Leukemia Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 888-888 ◽  
Author(s):  
Okinaka Keiji ◽  
Satoki Nakamura ◽  
Isao Hirano ◽  
Takaaki Ono ◽  
Shinya Fujisawa ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Lines ◽  
Leukemia Cell ◽  
Aurora Kinase ◽  
Leukemia Cells ◽  
Clinical Samples ◽  
Acute Leukemias ◽  
Aurora Kinase B ◽  
Leukemia Cell Lines ◽  
Foxm1 Expression

Abstract [Background] FoxM1, a member of the Fox transcription factor family, plays an important cell cycle regulator of both the transition from G1 to S phase and progression to mitosis. FoxM1 expression was also found to be up-regulated in some solid tumors (basal cell carcinomas, hepatocellular carcinoma, and primary breast cancer). These results suggested that FoxM1 plays a role in the oncogenesis of malignancies. However, it is unknown whether FoxM1 expression contributes to the development or progression of leukemia cells. Therefore, we investigated how FoxM1 regulated the cell cycle of leukemia cells and the expression analysis of the FoxM1 gene in patients with acute leukemias. [Methods] The cells used in this study were human acute leukemia cell lines, U937 and YRK2 cells. Primary acute myeloblastic (25 AML (4 M1, 11 M2, 6 M4, 4 M5)) cells were obtained from the peripheral blood. Human normal mononuclear cells (MNCs) were isolated from peripheral blood (PB) of healthy volunteers after obtaining informed consents. For analysis of proliferation and mitotic regulatory proteins (p27, p21, Skp2, Cdc25B, Cyclin D1, Survivin, Aurora kinase B, and KIS) in leukemia cells, MTT assays and western blot were performed in all cell lines, which untransfected or transfected with siRNA FoxM1, respectively. For cell cycle analysis, flow cytometory analysis was performed in leukemia cells untransfected or transfected with siRNAFoxM1 by PI staining. For analysis of FoxM1 mRNA, quantitative RT-PCR was performed in all cell lines and clinical samples. [Results] In all leukemia cell lines, the expression of FoxM1B mRNA were significantly higher than normal MNCs. When transfected with the siRNA FoxM1 in leukemia cells, suppression of FoxM1 caused a mean 71% (range 62 to 80%) reduction in S phase cells and a mean 4.4-fold (range 3.2 to 5.6-fold) increase in G2/M phase cells compared to controls. MTT assay demonstrated that the proliferation of the siRNA FoxM1 transfected cells was inhibited compared to the untransfected cells. Moreover, FoxM1 knockdown by siRNA in leukemia cells reduced protein and mRNA expression of Aurora kinase B, Survivin, Cyclin D1, Skp2 and Cdc25B, while increased protein expression of p21and p27. In the clinical samples obtained from patients with acute leukemias, the FoxM1B gene was overexpressed in 22/25 (88%). The relative folds of FoxM1B gene expression were for AML: 2.83 compared to normal MNCs. [Conclusions] In this study, we report in the first time that FoxM1 is overexpressed in myeloid leukemia cells. These results demonstrated that expression of FoxM1 is an essential transcription factor for growth of leukemia cells, and regulate expression of the mitotic regulators. Moreover, we showed that FoxM1 induced the expression of KIS protein. Therefore, FoxM1 might be one of moleculer targets of therapy for acute leukemias.

Rapamycin Causes Reversible Cell Cycle Slowing, Rather Than Cell Cycle Arrest, in Myeloid and Lymphoid Cell Lines and in Acute Leukemia Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5021-5021
Author(s):  
Amy Kimball ◽  
Mehmet Burcu ◽  
Kieran L O’Loughlin ◽  
Laurie A. Ford ◽  
Hans Minderman ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Acute Leukemia ◽  
Cell Cycle Arrest ◽  
Cell Lines ◽  
Lymphoid Cell ◽  
Leukemia Cells ◽  
Time Points ◽  
Cycle Arrest ◽  
Doubling Times ◽  
Lymphoid Cell Lines

Abstract The mammalian target of rapamycin (mTOR), a serine/threonine protein kinase that regulates cell survival and proliferation, is constitutively activated in diverse malignancies, and constitutive mTOR activation has been implicated in both malignant transformation and resistance to chemotherapy. The mTOR inhibitor rapamycin, which inhibits mTOR signaling and has immunosuppressive effects at nanomolar concentrations, also has antineoplastic activity, but its reported effects on cell cycle and on apoptosis have been variable. Several studies evaluated lymphoid and myeloid cell lines after 24 or 48 hours of exposure to rapamycin and reported cell cycle arrest in G1, while others reported apoptosis. Rapamycin is being incorporated into treatment regimens in acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and other hematologic malignancies, and knowledge of its growth inhibitory and cytotoxic effects will be useful in designing combination regimens. To this end, the concentration-dependent effects of rapamycin on survival, proliferation, cell cycle and apoptosis of myeloid and lymphoid cell lines and acute leukemia cells were studied. Assays were performed at serial time points extending to 96 hours. Cells studied included HL60 myeloid and Jurkat T-lymphoblastic leukemia, 8226 myeloma and U937 histiocytic lymphoma cells and ML-1 myeloid leukemia cells, which have intact p53, as well as multidrug resistant HL60/VCR, HL60/ADR and 8226/MR20 cells overexpressing the ATP-binding cassette (ABC) proteins ABCB1, ABCC1 and ABCG2, respectively. Pretreatment blasts from 9 patients with AML and 4 with ALL were also studied. Cell survival was measured following 96-hour (cell lines) or 48-hour (patient samples) culture with rapamycin at picomolar to micromolar concentrations using the WST-1 colorimetric assay. Cells grown in suspension culture without rapamycin and with 1 nanomolar to 100 micromolar rapamycin were counted at serial time points. Cell cycle was analyzed by propidium iodide labeling and bromodeoxyuridine incorporation, cell division by carboxyfluorescein succinimidyl ester (CFSE) staining and apoptosis by Annexin V labeling, all measured by flow cytometry. Concentration-dependent effects of rapamycin on cell lines and acute leukemia cells in the WST-1 assay were biphasic, occurring at nanomolar and again at micromolar concentrations, with a plateau from approximately 10 nanomolar to 10 micromolar. At nanomolar concentrations rapamycin prolonged cell doubling times by 20–40%, as evidenced by CFSE staining at serial time points, but it did not cause cell cycle arrest. Examined at serial time points, cells continuously exposed to rapamycin at nanomolar concentrations did not accumulate in any single phase of the cell cycle, nor did they become apoptotic. Doubling times increased, resulting in decreased cell numbers at each time point in relation to control cultures without rapamycin. When cells cultured with rapamycin were transferred to rapamycin-free medium, doubling times reverted to those in rapamycin-free cultures, demonstrating that cell cycle slowing resulted from continuous exposure to rapamycin and was fully reversible in the absence of rapamycin. In contrast to rapamycin at nanomolar concentrations, rapamycin at micromolar concentrations caused apoptosis of cell lines and acute leukemia cells. Thus rapamycin at nanomolar concentrations increases the doubling time of myeloid and lymphoid cell lines and acute leukemia cells, while rapamycin at micromolar concentrations causes apoptosis. Rapamycin doses used in immunosuppressive regimens are well tolerated and yield nanomolar plasma concentrations. Our data suggest that continuous oral administration of rapamycin or its analogs at the doses used in immunosuppressive regimens may slow growth of leukemia cells and might have utility as maintenance therapy in the minimal residual disease setting, given the known lack of effect on normal cells. Higher doses of rapamycin are being studied in phase I clinical trials. Finally, increased understanding of rapamycin’s effect on leukemia cell growth will help direct its incorporation into combination therapy.

Bortezomib Interacts Synergistically with Belinostat to Induce Apoptosis In Human Acute Myeloid and Lymphoid Leukemia Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3266-3266
Author(s):  
Yun Dai ◽  
Shuang Chen ◽  
Li Wang ◽  
Xin-Yan Pei ◽  
Lora Kramer ◽  
...  
Keyword(s):  
Acute Leukemia ◽  
Cell Lines ◽  
Leukemia Cell ◽  
Dose Effect ◽  
Leukemia Cells ◽  
Parp Cleavage ◽  
Lymphoid Leukemia ◽  
Acute Leukemias ◽  
Leukemia Cell Lines ◽  
Acute Myeloid

Abstract Abstract 3266 Previous studies have demonstrated interactions between histone deacetylase (HDAC) and proteasome inhibitors (PIs) in multiple myeloma, NHL, and CLL. However, exploration of this strategy in acute leukemias has been more limited. In this context, we have previously demonstrated that HDACIs activate the cytoprotective NF-κB pathway in acute myeloid leukemia (AML) cells, and that interruption of this process dramatically increases lethality. Such findings raise the possibility that PIs, which block degradation of the NF-κB-inhibitory protein IκBα, may act via an analogous mechanism in acute leukemias. Consequently, interactions between the clinically relevant pan-HDAC inhibitor belinostat (PXD-101) and the FDA-approved proteasome inhibitor bortezomib were evaluated in both continuously cultured cell lines and primary AML and acute lymphoid leukemia (ALL) samples. First, whereas each agent individually displayed only modest toxicity, co-treatment for 24 hr or 48 hr with low concentrations of bortezomib (3 - 5 nM) and belinostat (50 - 300 nM) led to pronounced increases in apoptosis in diverse human acute leukemia cell lines (e.g., AML, U937, HL-60, MV-4-11/Flt3-ITD; T-cell ALL, Jurkat; B-cell ALL, SEM). Interactions between these agents were determined to be synergistic by Median Dose Effect analysis. Significantly, equivalent interactions were observed in multiple primary AML (n = 4) and ALL (n = 3) blast specimens, while largely sparing normal CD34+ hematopoietic cells isolated from umbilical cord blood (n = 4), as determined by annexin V/PI, DiOC6, and/or 7-AAD uptake by flow cytometry. Western blot analysis demonstrated that co-exposure of primary leukemia blasts to bortezomib and belinostat resulted in marked increase in PARP cleavage, compared with each agent administrated alone. In addition, cell morphology exhibited classical features of apoptosis in primary acute leukemia blasts, but not in normal CD34+ cells, following combination treatment. Second, in both cell lines and primary blasts, administration of bortezomib resulted in accumulation of the phosphorylated (S32/S36) form of IκBα, accompanied by diminished belinostat-mediated hyperacetylation (K310) of RelA/p65. Bortezomib also blocked processing of the precursor p100 into the active p52, an event enhanced by co-treatment with belinostat. These results indicate that a regimen combining bortezomib and belinostat interrupts both canonical and non-canonical NF-κB signaling pathways in acute leukemia cells. Moreover, co-exposure to these agents diminished expression of NF-κB-dependent pro-survival proteins including Bcl-xL, XIAP, and SOD2, but not NF-κB-independent anti-apoptotic proteins such as survivin. Third, because the BH3-only Bcl-2 family pro-apoptotic protein Bim plays an important role in the lethality of PIs or HDACIs as single agents, the expression and functional role of Bim in bortezomib/belinostat interactions was examined. Notably, whereas treatment with marginally toxic concentrations of either agent alone clearly increased Bim protein levels, co-exposure of either leukemia cell lines or primary blasts to bortezomib and belinostat led to sharply increased Bim expression (particularly the BimEL isoform). Importantly, shRNA knock-down of Bim substantially attenuated lethality mediated by co-treatment with bortezomib and belinostat in both AML (U937) and ALL (Jurkat) cells, supporting the notion that up-regulation of Bim plays a critical role in anti-leukemic activity of the combination regimen. Lastly, exposure of cultured leukemia cells and primary blasts to belinostat ± bortezomib induced hyperacetylation of a-tubulin, indicating inhibition of HDAC6, a microtubule-associated deacetylase that regulates aggresome formation and cell survival in response to misfolded protein-induced stress. Together, these findings indicate that the regimen combining belinostat and bortezomib is highly active against human AML and ALL cells, including primary leukemic blasts, in association with perturbation in the balance between pro-survival (NF-κB-dependent) and pro-death (e.g., Bim) signals. They also suggest that this strategy warrants further attention in acute leukemias. Accordingly, a Phase I trial of belinostat and bortezomib in patients with refractory acute leukemia or MDS has recently been initiated. Disclosures: Off Label Use: Investigational use of belinostat and bortezomib.

scholarly journals Pre-Clinical Evaluation of the Proteasome Inhibitor Ixazomib against Bortezomib-Resistant Leukemia Cells and Primary Acute Leukemia Cells

Cells ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 665
Author(s):  
Margot S.F. Roeten ◽  
Johan van Meerloo ◽  
Zinia J. Kwidama ◽  
Giovanna ter Huizen ◽  
Wouter H. Segerink ◽  
...  
Keyword(s):  
Acute Leukemia ◽  
Cell Lines ◽  
Proteasome Inhibitor ◽  
Ex Vivo ◽  
Lymphoblastic Leukemia ◽  
Unmet Need ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Cross Resistance ◽  
Proteasome Subunit

At present, 20–30% of children with acute leukemia still relapse from current chemotherapy protocols, underscoring the unmet need for new treatment options, such as proteasome inhibition. Ixazomib (IXA) is an orally available proteasome inhibitor, with an improved safety profile compared to Bortezomib (BTZ). The mechanism of action (proteasome subunit inhibition, apoptosis induction) and growth inhibitory potential of IXA vs. BTZ were tested in vitro in human (BTZ-resistant) leukemia cell lines. Ex vivo activity of IXA vs. BTZ was analyzed in 15 acute lymphoblastic leukemia (ALL) and 9 acute myeloid leukemia (AML) primary pediatric patient samples. BTZ demonstrated more potent inhibitory effects on constitutive β5 and immunoproteasome β5i proteasome subunit activity; however, IXA more potently inhibited β1i subunit than BTZ (70% vs. 29% at 2.5 nM). In ALL/AML cell lines, IXA conveyed 50% growth inhibition at low nanomolar concentrations, but was ~10-fold less potent than BTZ. BTZ-resistant cells (150–160 fold) displayed similar (100-fold) cross-resistance to IXA. Finally, IXA and BTZ exhibited anti-leukemic effects for primary ex vivo ALL and AML cells; mean LC50 (nM) for IXA: 24 ± 11 and 30 ± 8, respectively, and mean LC50 for BTZ: 4.5 ± 1 and 11 ± 4, respectively. IXA has overlapping mechanisms of action with BTZ and showed anti-leukemic activity in primary leukemic cells, encouraging further pre-clinical in vivo evaluation.

scholarly journals Second Allogeneic Hematopoietic Stem Cell Transplantation for Post-Transplant Relapsed Acute Leukemia in Children - an EBMT PDWP Retrospective Study

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 912-912
Author(s):  
Isaac Yaniv ◽  
Aviva C. Krauss ◽  
Eric Beohou ◽  
Arnaud Dalissier ◽  
Selim Corbacioglu ◽  
...  
Keyword(s):  
Prognostic Factors ◽  
Prognostic Factor ◽  
Acute Leukemia ◽  
Pediatric Patients ◽  
Multivariate Analyses ◽  
Acute Leukemias ◽  
Hematopoietic Stem ◽  
Entire Cohort

Abstract Introduction Using the EBMT registry, we retrospectively analyzed outcomes for 373 pediatric patients who underwent second allogeneic transplant for relapsed acute leukemia at 120 centers in 32 countries, between the years 2004 and 2013, in an attempt to assess relapse, survival, GVHD and other outcomes, as well as identify factors correlating with prognosis in this cohort of patients. To our knowledge, this is the largest analysis of pediatric patients undergoing second allogeneic HSCT for relapsed acute leukemia to date. This allowed for an independent analysis of each disease, including 214 patients with ALL and 159 with AML. Patients and Methods Centers received a questionnaire completing data already available in the ProMISe database on patients between 0-18 years of age treated between 2004 and 2013. Results A total of 387 patients received a second SCT after relapse. 373 have been included in the analysis, 214 for ALL and 159 for AML. Detailed data were available for 201 patients from 48 centers; for the remainder, analysis was based on the registry. For the entire cohort overall survival (OS) at 2 and 5 years were 38% and 29%, and leukemia free survival (LFS) 30% and 25% respectively. ALL: With a median follow up from 2nd SCT of 36.4 months, OS at 1 and 5 years were 47% and 28% respectively. LFS was 39% and 28% respectively. NRM at 2 years was 22%. In multivariate analyses favorable prognostic factors for both OS and LFS were: CR prior to 2nd SCT (p=0.0001), interval > 12 months between transplants (p=0.0007), use of myeloablative conditioning (p=0.039) and the presence of cGvHD after the first SCT (p=0.0001). Good prognostic factor for low NRM was interval of more than 12 months between transplants (p=0.0002). AML: With a median follow up from 2nd SCT of 50 months, OS at 1 and 5 years were 44% and 15% respectively. LFS was 28% and 15% respectively. NRM at 2 years was 18%. In multivariate analyses, favorable prognostic factors for OS as well as LFS were: CR prior to 2nd SCT (p=0.031;0.044 respectively), interval > 6 months between transplants (p=0.0003;0.0001 respectively), and having cGvHD after the first SCT (p=0.0001). Most patients experience disease relapse or NRM within the first year after their second transplant. This observation seems to be more consistent in patients transplanted for ALL, with more changes over time in patients with AML. For ALL in particular, the 2-year incidences of relapse, NRM and LFS were not different from those at 5-years. Even in the relapse setting, survival rates for patients with ALL remain superior to patients with AML, consistent with the prognostic differences at diagnosis. Our findings, consistent for the AML and ALL subgroups, suggest that cGHVD prior to second HSCT is associated with better outcome. The identification of cGHVD prior to second transplant has not been heretofore described as a favorable prognostic factor. This strong correlation merits further study, specifically as to the underlying biology for this association. Conclusion Children with relapsed acute leukemias have a substantial chance to become long term survivors following a second SCT. CR prior to second SCT, longer interval between transplants and the presence of cGvHD after the first transplant, are favorable prognostic factors for ALL and AML. Our findings may help physicians in discussing the risk-benefit of a second transplant. These results are particularly relevant in an era where an explosion of new therapies, specifically targeted therapies and those that modulate the immune response, behoove us to carefully identify subpopulations of patients for whom specific therapies are appropriate. Novel approaches are needed to minimize relapse risk as well as short and long term morbidity in these pediatric patients while considering a second SCT for relapsed acute leukemia. Disclosures Corbacioglu: Jazz Pharmaceuticals: Consultancy, Honoraria. Bader: Novartis, Medac, Amgen, Riemser, Neovii: Consultancy, Honoraria, Research Funding.

scholarly journals The chromosome 4q21 gene (AF-4/FEL) is widely expressed in normal tissues and shows breakpoint diversity in t(4;11)(q21;q23) acute leukemia

Blood ◽  
1993 ◽  
Vol 82 (4) ◽  
pp. 1080-1085 ◽  
Author(s):  
CS Chen ◽  
JM Hilden ◽  
J Frestedt ◽  
PH Domer ◽  
R Moore ◽  
...  
Keyword(s):  
Acute Leukemia ◽  
Cell Lines ◽  
Chromosomal Translocation ◽  
Leukemic Cell ◽  
Genomic Region ◽  
Acute Leukemias ◽  
Normal Tissues ◽  
Leukemic Cell Lines ◽  
A Minor ◽  
Chromosome 4Q21

The chromosomal translocation, t(4;11)(q21;q23), is the most common type of 11q23 chromosomal abnormality, being highly prevalent in infant acute leukemias and associated with a poor prognosis. The t(4;11) results in the fusion of an 11q23 gene (MLL, HRX, Htrx-1, or ALL-1) and a 4q21 gene (AF-4 or FEL). To further evaluate the 4q21 gene and its role in t(4;11) acute leukemia, we have cloned a 38-kb genomic region and mapped exons of the AF-4 gene. The 4q21 breakpoints in 19 cases of t(4;11) acute leukemia were analyzed by Southern analysis and pulsed- field gels. Seventeen of the 19 cases had breakpoints on chromosome 4q21 that were scattered in this 38 kb region. Expression of the AF-4 gene was studied in a total of 28 various nonhematopoietic, hematopoietic, and t(4;11) leukemic cell lines. The AF-4 gene was expressed in all cell lines as a major and a minor transcript. In addition to the normal transcripts, two fusion transcripts from the derivative 11 and derivative 4 chromosomes were identified in all t(4;11) cell lines except B1, which had only the der(11) transcript. These findings suggest that the breakpoints on 4q21 cluster over a broader area than do the breakpoints in the 11q23 gene, and that der(11) encodes the fusion RNA found consistently in leukemia cells.

scholarly journals A Gain of Function Mutation in the NSD2 Histone Methyltransferase Drives Glucocorticoid Resistance Via Blocking Receptor Auto-Induction and BIM/Bmf Expression in ALL

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3758-3758
Author(s):  
Jianping Li ◽  
Catalina Troche ◽  
Julia Hlavka Zhang ◽  
Jonathan Shrimp ◽  
Jacob S. Roth ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Line ◽  
Cell Lines ◽  
Mutant Allele ◽  
Gene Editing ◽  
Conflicts Of Interest ◽  
Chemotherapeutic Agents ◽  
Rna Seq ◽  
Mesenchymal Transition ◽  
Pediatric All

Despite improvements in chemotherapy that have increased the 5-year survival rates of pediatric ALL to close to 90%, 15-20% of patients may relapse with a very poor prognosis. Pediatric ALL patients, particularly those in relapse can harbor a specific point mutation (E1099K) in NSD2 (nuclear receptor binding SET domain protein 2) gene, also known as MMSET or WHSC1, which encodes a histone methyl transferase specific for H3K36me2. To understand the biology of mutant NSD2, we used CRISPR-Cas9 gene editing to disrupt the NSD2E1099K mutant allele in B-ALL cell lines (RCH-ACV and SEM) and T-ALL cell line (RPMI-8402) or insert the E1099K mutation into the NSD2WT T-ALL cell line (CEM) and B-ALL cell line (697). Cell lines in which the NSD2E1099K mutant allele is present display increased global levels of H3K36me2 and decreased H3K27me3. NSD2E1099Kcells demonstrate enhanced cell growth, colony formation and migration. NSD2E1099K mutant cell lines assayed by RNA-Seq exhibit an aberrant gene signature, mostly representing gene activation, with activation of signaling pathways, genes implicated in the epithelial mesenchymal transition and prominent expression of neural genes not generally found in hematopoietic tissues. Accordingly, NSD2E1099K cell lines showed prominent tropism to the central neural system in xenografts. To understand why this NSD2 mutations are identified prominently in children who relapse early from therapy for ALL, we performed high-throughput screening in our isogenic cell lines with the National Center for Advancing Translation Science (NCATS) Pharmaceutical Collection and other annotated chemical libraries and found that NSD2E1099K cells are resistant to glucocorticoids (GC) but not to other chemotherapeutic agents used to treat ALL such as vincristine, doxorubicin, cyclophosphamide, methotrexate, and 6-mercaptopurine. Accordingly, patient-derived-xenograft ALL cells with NSD2E1099K mutation were resistant to GC treatment. Reversion of NSD2E1099K mutation to NSD2WT restored GC sensitivity to both B- and T-ALL cell lines, which was accompanied by cell cycle arrest in G1 and induced-apoptosis. Furthermore, knock-in of the NSD2E1099K mutation conferred GC resistance to ALL cell lines by triggering cell cycle progression, proliferation and anti-apoptotic processes. Mice with NSD2E1099K xenografts were completely resistant to GC treatment while treatment of mice injected with isogenic NSD2WT cells led to significant tumor reduction and survival benefit. To illustrate these biological phenotypes and understand the molecular mechanism of GC resistance driven by NSD2E1099Kmutation, we investigated the GC-induced transcriptome, GC receptor (GR) binding sites and related epigenetic changes in isogenic ALL cell lines in response to GC treatment. RNA-Seq showed that GC transcriptional response was almost completely blocked in NSD2E1099K cells, especially in T-ALL cell lines, correlating with their lack of biological response. GC treatment activated apoptotic pathways and downregulated cell cycle and DNA repair pathways only in NSD2WT cells. The critical pro-apoptotic regulators BIM and BMF failed to be activated by GC in NSD2E1099K cells but were prominently activated when the NSD2 mutation was removed. Chromatin immunoprecipitation sequencing (ChIP-Seq) showed that, the NSD2E1099K mutation blocked the ability of GR and CTCF to bind most GC response elements (GREs) such as those within BIM and BMF. While GR binding in NSD2WT cells was accompanied by increased H3K27 acetylation and gene expression, this failed to occur in NSD2 mutant cells. Furthermore, we found that GR RNA and protein levels were repressed in ALL cells expressing NSD2E1099K and GC failed to induce GR expression in these cells. Paradoxically, while H3K27me3 levels were generally decreased in NSD2E1099K cells, we saw increased levels of H3K27me3 at the GRE within the GR gene body where GR itself and CTCF normally bind, suggesting a novel role for the polycomb repressive complex 2 and EZH2 inhibitors for this form of GC resistance. In conclusion, these studies demonstrate that NSD2E1099K mutation may play an important role in treatment failure of pediatric ALL relapse by interfering with the GR expression and its ability to bind and activate key target genes. Gene editing screens are being performed to understand how to overcome this resistance. Disclosures No relevant conflicts of interest to declare.

Analysis of Aurora Kinase Expressions and Cell Cycle Regulation by Aurora-C in Leukemia Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1366-1366 ◽  
Author(s):  
Miki Kobayashi ◽  
Satoki Nakamura ◽  
Takaaki Ono ◽  
Yuya Sugimoto ◽  
Naohi Sahara ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Western Blot ◽  
Cell Lines ◽  
Mammalian Cells ◽  
Cell Cycle Progression ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Human Leukemia ◽  
Aurora A ◽  
Aurora Kinases

Abstract Background: The conserved Aurora family kinases, a family of mitotic serine/threonine kinases, have three members (Aurora-A, -B and -C) in mammalian cells. The Aurora kinases are involved in the regulation of cell cycle progression, and alterations in their expression have been shown to associate with cell malignant transformation. Aurora A localizes to the centrosomes during anaphase, and it is required for mitotic entry. Aurora B regulates the formation of a stable bipolar spindle-kinetochore attachment in mitosis. The function of Aurora-C in mammalian cells has not been studied extensively. In this study, we investigated that human leukemia cells expressed all 3 Aurora kinases at both protein and mRNA level, and the mechanisms of cell cycle regulation by knock down of Aurora C in leukemia cells. Methods: In this study, we used the 7 human leukemia cell lines, K562, NB4, HL60, U937, CEM, MOLT4, SUP-B15 cells. The expression levels of mRNA and proteins of Aurora kinases were evaluated by RT-PCR and western blot. The analysis of proliferation and cell cycle were performed by MTT assay and FCM, respectively. Results: The mRNA of Aurora-A and Aurora-B are highly expressed in human leukemia cell lines (K562, NB4, HL60, U937, CEM, MOLT4, SUP-B15 cells), while the mRNA of Aurora C is not only expressed highly in all cells. In contrast, an increase in the protein level of the 3 kinases was found in all cell lines. These observations suggested posttranscriptional mechanisms, which modulate the expression of Aurora C. In cell cycle analysis by flow cytometory, the knock down of Aurora C by siRNA induced G0/G1 arrest and apoptosis in leukemia cells, and increased the protein levels of p27Kip1 and decreased Skp2 by western blot. In MTT assay, it was revealed that the growth inhibition of leukemia cells transfected with siRNA Aurora C compared with leukemia cells untransfected with siRNA Aurora C. Moreover, We showed that Aurora C was associated with Survivin and directly bound to Survivin by immunoprecipitation and western blot. Conclusion: We found that human leukemia cells expressed all 3 members of the Aurora kinase family. These results suggest that the Aurora kinases may play a relevant role in leukemia cells. Among these Aurora kinases, Aurora C interacted with Survivin and prevented apoptosis of leukemia cells, and induced cell cycle progression. Our results showed that Aurora-C may serve as a key regulator in cell division and survival. These results suggest that the Aurora C kinase may play an important role in leukemia cells, and may represent a target for leukemia therapy.

Synergistic Apoptosis Induction by Betulinic Acid and Established Cytotoxic Drugs in Leukemia Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3096-3096 ◽  
Author(s):  
Harald Ehrhardt ◽  
Ines Höfig ◽  
Irmela Jeremias
Keyword(s):  
Acute Leukemia ◽  
Tumor Cells ◽  
Cell Lines ◽  
Solid Tumor ◽  
Betulinic Acid ◽  
Cytotoxic Drugs ◽  
Leukemia Cells ◽  
Cancer Drug ◽  
Apoptosis Induction ◽  
Apoptosis Resistance

Abstract Abstract 3096 Poster Board III-33 Introduction Betulinic acid (BA) represents an effective inducer of apoptosis in a broad spectrum of solid tumor cells in vitro and in small animal models in vivo. Due to its low toxicity in animal trials, it represents a putative future anti-cancer drug. We first described that in addition to solid tumor cells, BA potently induces apoptosis in leukemia cells expanding the therapeutic use of BA to hematological malignancies (Ehrhardt et al., Leukemia 2004). Purpose Here we asked how BA might best be incorporated into polychemotherapy protocols used to treat acute leukemia and therefore searched for conventional cytotoxic drugs which enhance the anti-tumor effect of BA. Of suitable drugs discovered, we characterized the molecular mechanisms determining the synergistic interaction with BA. Methods We used both leukemia cell lines and primary tumor cells obtained from children with acute lymphoblastic or myeloid leukemia at diagnosis of disease or relapse. Primary, patient-derived tumor cells were further amplified in NOD/SCID mice. Most importantly, we transfected primary, patient-derived tumor cells to knock down endogenous apoptosis signaling proteins. Results – phenotype When conventional cytotoxic drugs in routine use to treat acute leukemia were screened on leukemic cell lines, three drugs were identified which induce synergistic induction when given together with BA: doxorubicin, asparaginase and vincristine. Accordingly, clonogenic survival was reduced in a super-additive way, when one of these drugs was combined with BA. Importantly, synergistic apoptosis induction by these drugs with BA was also found in primary, patient-derived leukemic tumor samples. Both in primary samples and cell lines, BA-induced apoptosis was enhanced by addition of the second drug, even if doxorubicin, asparaginase or vincristine alone were unable to induce apoptosis due to apoptosis resistance. Results – mechanism Synergistic apoptosis induction was accompanied by increased caspase activation and was inhibited by the addition of zVAD, by overexpression of XIAP or knockdown of Caspase-9. p53 was activated nearly exclusively, when doxorubicin, asparaginase or vincristine was combined with BA and knockdown of p53 inhibited synergistic apoptosis induction of the drug combinations. While expression of Bak, Bim, Bid, Bcl-2, Bcl-xL and PUMA remained unchanged by stimulation with BA and doxorubicin, asparaginase or vincristine, the p53 target gene NOXA was strongly upregulated exclusively when drugs were combined. When doxorubicin, asparaginase or vincristine were given together with BA, more apoptogenic factors like Smac and Cytochrome c were released from mitochondria and synergistic apoptosis induction by BA with either doxorubicin, asparaginase or vincristine depended on increased mitochondrial signaling. Knockdown of Bim, Bid or PUMA did not alter synergistic apoptosis induction by BA and doxorubicin, asparaginase or vincristine. In contrast, overexpression of Bcl-2 or Bcl-xL or knockdown of either Bak or NOXA inhibited synergistic apoptosis induction. Knockdown of p53 and NOXA in primary, patient-derived leukemia cells completely inhibited synergistic apoptosis induction by BA and doxorubicin, asparaginase or vincristine. Conclusion Our data show that BA induces synergistic apoptosis induction when given in combination with doxorubicin, asparaginase and vincristine based on increased activation of p53 which enables expression of NOXA and a NOXA – Bak dependent activation of mitochondria. BA should best be incorporated into future anti-leukemia polychemotherapy protocols in close proximity to doxorubicin, asparaginase or vincristine. Disclosures No relevant conflicts of interest to declare.

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