The Acrylonitrile Analog, VJ-289 Ablates Acute Myelogenous Leukemia Blast, Progenitor and Stem Cell Populations by Inducing Tubulin Acetylation and Caspase Activation

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2496-2496
Author(s):  
Hongliang Zong ◽  
Narsimha Reddy Penthala ◽  
Siddhartha Sen ◽  
Sarah Brennan ◽  
Vijayakumar Sonar ◽  
...  
Keyword(s):  
Cell Lines ◽  
Leukemia Cell ◽  
Leukemic Cell ◽  
Dependent Manner ◽  
Caspase Activation ◽  
Tubulin Acetylation ◽  
Leukemia Cell Lines ◽  
Cord Blood Cells ◽  
Stem Cell Populations ◽  
Dose Dependent

Abstract Abstract 2496 Combretastatin A-4, a derivative of combretastatin, a natural product of the South African tree Combretum caffrum, has been reported to have anti-angiogenic and anti-tubulin effects in different cancer cell lines. We synthesized 48 novel combretastatin analogs to assess anti-leukemia activity in a panel of 12 leukemia cell lines. We identified an analog, VJ-289 [(Z)-3-(1H-indol-2-yl)-2-(3,4,5-trimethoxyphenyl)acrylonitrile] with robust anti-leukemic activity. VJ-289 showed a dose-dependent toxicity to most of the leukemic cell lines tested. The average LD50 for the 12 different leukemia cell lines was 132 nM (95% CI, 91.8–170.5). Specifically, MV4-11 cells demonstrated the most sensitivity to VJ-289 (LD50 = 66 nM), whereas THP-1 was the most resistant (LD50 = 227 nM). Furthermore, when the activity of VJ-289 was tested, five out of 14 primary AML samples demonstrated resistance to VJ-289 with an LD50 > 300nM. The average LD50 for the sensitive primary AML samples was 64.06 nM (95% CI, 35.36–92.76; N=9). Most importantly, normal CD34+ cord blood cells were significantly less affected by VJ-289 (LD50 > 500 nM). Furthermore, VJ-289 was capable of eliminating AML progenitor/stem cells as determined by phenotypic analysis in 15 primary AML samples, colonies forming ability (N=6) and xenotransplant assays (N=6). Overall, we observed a 90.3% decrease in colony formation after treatment with 150 nM VJ-289 relative to untreated control. In contrast, VJ-289 had less impact on colony forming ability of normal hematopoietic stem/progenitor cells from cord blood cells (66.1% decrease relative to untreated; p=0.013). To investigate the role of VJ-289 in leukemic cell apoptosis, various cell survival signaling pathways were examined. Western blotting and intracellular staining/flow cytometry data showed that caspases, including caspase 3 and 8, were activated alongside the cleavage of PARP in a dose-dependent manner. Caspase activation was observed as early as 4 h after treatment with 100 nM VJ-289. PI3K/AKT, MAPK and NF-κB were decreased upon VJ-289 treatment. Moreover, the degradation of MCL1 and the cleavage of Bcl2, two anti-apoptotic Bcl2 family members, were decreased by VJ-289 in a dose- and time-dependent manner. Interestingly, the acetylation of α-tubulin, which is critical for microtubule stabilization, and is involved in multiple cellular functions, ranging from cell motility, cell cycle progression or cell differentiation to intracellular trafficking and signaling, was transiently induced by VJ-289 within 2 hours, and was inhibited dramatically after 4 hours. In summary, we have identified a combrestastatin A-4 analog, VJ-289, as a new anti-leukemia agent with the ability to ablate blast, progenitor and stem cell populations via induction of caspase activation and α-tubulin acetylation. Studies are underway to determine what modulates sensitivity to VJ-289 across AML specimens. Disclosures: No relevant conflicts of interest to declare.

Dysregulated Expression of HSP70 Isoforms in Acute Myelogenous Leukemia (AML)

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1370-1370
Author(s):  
Krishan K. Sharma ◽  
Juan Felipe Rico ◽  
Duane C Hassane ◽  
Gabriela Chiosis ◽  
Monica L. Guzman
Keyword(s):  
Drug Resistance ◽  
Heat Shock ◽  
Cord Blood ◽  
Cell Lines ◽  
Blood Cells ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Normal Cells ◽  
Leukemia Cell Lines ◽  
Cord Blood Cells

Abstract Abstract 1370 Stress-inducible heat shock protein 70 (HSP70) is a major cytoprotective factor and a molecular chaperone that interacts with HSP90 to form a multi-chaperone complex. Cancer cells are highly dependent on this complex due to their increased demand for protein synthesis. HSP70 overexpression inhibits apoptosis and has been associated with drug resistance. However, the contribution to drug resistance in AML of specific HSP70 isoforms remains unknown. As there is growing interest in therapeutically targeting HSP70, we investigated the expression of 7 different HSP70 isoforms in AML primary cells and leukemia cell lines and their response to a novel HSP70-inhbitor, YK5. A panel of 12 leukemia cell lines and 11 primary samples was used to determine the expression of HSP70 and their response to YK5. We also evaluated the changes to the HSP70 isoforms when exposed to either heat shock or YK5. We found MV4-11, MOLM-13, and U937 sensitive to YK5 (LD50 = 1.18μM, 1.03μM, and 2.31μM at 24 hours, respectively). In contrast, OCI-AML3, TUR and THP-1 were more resistant to the inhibitor. (LD50 = 9.92μM, 9.74μM, and 8.84μM at 24 hours, respectively). Non-tumor cells, however, were significantly less affected by treatment with YK5 (72% viable cord blood mononuclear cells after 24 hour treatment with 5μM YK5). We found that the cell surface expression of HSP70 was higher in both cell lines and primary samples when compared their normal counterparts. Furthermore, quantitative PCR revealed that cell lines with higher levels of HSPA1A and lower levels of HSPA6 demonstrated higher sensitivity to YK5. Interestingly, higher levels of HSPA1A and lower levels of HSPA6 were also found in primary AML samples when compared to CD34+ cord blood cells, consistent with the relative insensitivity of normal cells to YK5. We further discovered, mining publicly available databases, that high levels of HSPA1A were associated poorer prognosis (p = 0.004), suggesting that YK5 would be beneficial to patients presenting high HSP70 expression. We also evaluated the effect of YK5 on the gene expression of the various HSP70 isoforms. Quantitative PCR revealed the ability of YK5 to downregulate HSPA6 and HSC70 (HSPA8) in both cell lines and primary samples. Strikingly, all HSP70 isoforms exhibited similar fold changes upon heat shock in primary samples, CD34+ cord blood cells, and leukemia cell lines, indicating that the cellular stress response is not damaged in AML. However, the specificity of HSP70 inhibition to leukemia cells and not normal cells suggests a dysregulated set of client proteins and increased dependency on HSP70 to maintain leukemic homeostasis. In summary, we have found dysregulated expression of the HSP70 isoforms HSPA1A and HSPA6 in leukemia cells and that the expression levels of these isoforms correlate to the sensitivity of YK5-mediated HSP70 inhibition (HSPA1A: p=0.0012 and r2=0.801, HSPA6: p=0.0011 and r2=0.847). *KKS and JFR contributed equally to this project Disclosures: No relevant conflicts of interest to declare.

scholarly journals Effect of tamoxifen on cell lines displaying the multidrug-resistant phenotype [see comments]

Blood ◽  
1991 ◽  
Vol 77 (4) ◽  
pp. 818-825 ◽  
Author(s):  
E Berman ◽  
M Adams ◽  
R Duigou-Osterndorf ◽  
L Godfrey ◽  
B Clarkson ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Leukemia Cell ◽  
Multidrug Resistant ◽  
Leukemia Cell Line ◽  
Parent Cell ◽  
Continuous Exposure ◽  
Dependent Manner ◽  
Uptake Pattern ◽  
Dose Dependent

Abstract We examined the effect of tamoxifen (Tmx), verapamil, and daunorubicin (DNR) in two cell lines that displayed the multidrug-resistant (MDR) phenotype and used laser flow cytometry to quantitate intracellular DNR content. In the vinblastine-resistant human lymphoblastic lymphoma cell line CEM-VBL, simultaneous incubation of DNR with Tmx 10 mumol/L or Tmx 50 mumol/L increased intracellular DNR fluorescence in a dose-dependent manner and demonstrated an uptake pattern similar to that seen with DNR and verapamil. Similar results were obtained in the vincristine- resistant human myeloid leukemia cell line HL-60/RV+. Cellular retention of DNR was also measured in both cell lines and results suggested that continuous exposure of the cells to Tmx resulted in higher intracellular DNR content compared with cells resuspended in fresh medium. No effect of Tmx or verapamil was observed in the drug- sensitive parent cell lines CEM or HL-60. Clonogenic experiments were then performed to determine whether Tmx was itself inhibitory to cell growth or whether Tmx potentiated DNR cytotoxicity. Tmx 10 mumol/L did not significantly inhibit either CEM-VBL or HL-60/RV+ cells after a 3- hour exposure followed by culture in methylcellulose. Tmx 50 mumol/L was significantly more inhibitory in both cell lines. However, cells that had been incubated with DNR and Tmx 10 mumol/L demonstrated a marked increment in growth inhibition compared with cells that had been incubated with DNR alone or Tmx 10 mumol/L alone. Based on the data presented here, we suggest that clinical testing of Tmx and DNR be pursued in the setting where MDR may play a role.

The CXCR4 Antagonist AMD3100 Has Dual Effects, Initially Enhancing and Later Inhibiting Survival and Proliferation, in Myeloid Leukemia Cells in Vitro.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1721-1721
Author(s):  
Ha-Yon Kim ◽  
Ji-Young Hwang ◽  
Seong-Woo Kim ◽  
Gak-Won Yun ◽  
Young-Joon Yang ◽  
...  
Keyword(s):  
Cell Lines ◽  
Myeloid Leukemia ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Dependent Manner ◽  
Cxcr4 Antagonist ◽  
Hematopoietic Stem ◽  
Leukemia Cell Lines ◽  
Number Of Cells

Abstract Abstract 1721 Poster Board I-747 AMD3100, a small bicyclam antagonist for chemokine receptor CXCR4, induces the peripheral mobilization of hematopoietic stem cells. It also induces the segregation of leukemia cells in the bone marrow microenvironment, which should enhance the chemosensitivity of the cells. Based on these observations, AMD3100 is being considered for clinical use. However, AMD3100 activates G-protein coupled with CXCR4 and acts as a partial CXCR4 agonist. In this study, we explored whether AMD3100 affects the proliferation and survival of myeloid leukemia cells. As demonstrated previously, both AMD3100 and T140, another CXCR4 antagonist, markedly inhibited stromal cell-derived factor-1 (SDF-1)-induced chemotaxis and induced the internalization of CXCR4 in myeloid leukemia cell lines (U937, HL-60, MO7e, KG1a, and K562 cells) and CD34+ primary human acute myeloid leukemia (AML) cells. SDF-1 alone did not stimulate the proliferation of these leukemia cells, nor did it rescue the cells from apoptosis induced by serum deprivation. By contrast, AMD3100, but not T140, stimulated the proliferation of all five leukemia cell lines and primary AML cells in a dose-dependent manner in serum-free conditions for up to 5 days (∼ 2-fold increases at a concentration of 10-5M), which was abrogated by pretreating the cells with pertussis toxin. AMD3100 binds to CXCR7, another SDF-1 receptor, and all of the cells examined in this study expressed CXCR4 on the cell surface to some extent. The proliferation-enhancing effects of AMD3100 were not changed by knocking-down CXCR7 using the siRNA technique, whereas knocking-down CXCR4 significantly delayed the enhanced proliferation induced by AMD3100. Neither AMD3100 nor T140 induced the phosphorylation of Akt, Stat3, MAPK p44/p42, or MAPK p38, which are involved in SDF-1 signaling. In extended cultures of these cells for up to 14 days, AMD3100, but not T140, induced a marked decrease in the number of cells, compared to the control, after incubation for 5-7 days. Adding SDF-1 at the beginning and middle of the incubation did not affect the early increase or later decrease in the number of cells. AMD3100 reduced the apoptosis of these cells to a modest degree over the first 5-7 days and then markedly increased it. Consistent with the proliferation assay, AMD3100 increased the number of leukemia cell colonies during the early period of the assay, while it markedly decreased the number and size of the colonies in the later period of the assay. In conclusion, AMD3100 exerts dual effects, initially enhancing and subsequently inhibiting the survival and proliferation, in myeloid leukemia cells in vitro. Disclosures No relevant conflicts of interest to declare.

MDM2 Inhibitor Nutlin-3a Triggers Autophagic Cell Death In Addition to Apoptosis In Leukemia Cell Lines with Wild-Type p53

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3300-3300
Author(s):  
Seshagiri Duvvuri ◽  
Vivian Ruvolo ◽  
Duncan H. Mak ◽  
Kensuke Kojima ◽  
Marina Konopleva ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Death ◽  
Western Blot ◽  
Cell Lines ◽  
Research Funding ◽  
Leukemia Cell ◽  
Annexin V ◽  
Dependent Manner ◽  
Autophagic Vacuoles ◽  
Leukemia Cell Lines

Abstract Abstract 3300 Background: Nutlin-3a is a small molecule inhibitor of MDM2 and has been shown to induce apoptosis and cell cycle arrest in various cancer models in a p53 dependent manner. Autophagy is a programmed cell death that can occur concurrently with apoptosis or in its absence. There is significant debate whether autophagy is a protective mechanism or a bona fide mechanism of cell death. While autophagy can function as tumor cell defense mechanism against cellular stress induced death, mutation/loss of alleles of certain genes regulating autophagy have been associated with development of cancer (e.g. Beclin-1 in breast cancer [Nature, 1999, 402: 672–676]). Multiple proteins involved in autophagy are transcriptional targets of p53 but Nutlin-3a has not been evaluated for its role in inducing autophagy. Here we present data suggesting that low dose Nutlin-3a induces autophagy in addition to apoptosis in leukemia cell lines in a p53 dependent manner. Methods and results: OCI-AML-3 cells (p53-WT) treated with Nutlin-3a (2.5 and 5.0μM for 48, 72 and 96 hrs) were stained with mono-dansyl-cadaverine (MDC), a dye that accumulates in acidic autophagic vacuoles. OCI-AML-3 cells showed increasing staining with MDC in a dose and time dependent fashion by both flow cytometry (54%, 57% and 51% MDC positive after treatment with Nutlin-3a 5.0μM for 48, 72 and 96 hrs) and by confocal microscopy. Nutlin-3a treated cells also were positive for Annexin-V (flow cytometry 22%, 26% and 36% at 48, 72 and 96 hrs time points), and some of the cells were double-positive for Annexin-V and MDC (9.2%, 5% and 7% at 48, 72 and 96 hrs) suggesting that both apoptosis and autophagy can occur simultaneously. Autophagy induction was confirmed by Transmission Electron Microscopy (TEM). Large, multiple autophagic vacuoles were observed in OCI-AML-3 cells treated with Nutlin-3a. OCI-AML-3 cells with stable p53 knockdown by shRNA or HL-60 cells (p53-null) did not show increased MDC staining by flow cytometry (both cell lines) or autophagic vacuoles by TEM (HL-60) after similar treatment. Western blot analysis showed increases in LC3-II and in conjugation of Atg5/12, early and late autophagy markers respectively, in OCI-AML-3 cells after treatment with Nutlin-3a. Increased expression of the autophagy markers (LC3-II and Atg 5/12 conjugate) were also seen by Western blot analysis in the ALL cell lines REH and NALM-6 (both p53-WT) after treatment with Nutlin-3a. Western blot and/or RT-PCR analysis showed upregulation of other p53 related proteins involved in autophagy e.g. DRAM, AMPK-β, LKB1, pLKB1 in OCI-AML-3 cells treated with Nutlin-3a. As mTOR/Akt pathway inhibits autophagy, analysis of mTOR targets showed downregulation of the total and phospho-ribosomal-S6-protein levels, whereas there was no change in total or phospho-4-EBP-1 levels. Knockdown of Beclin-1 (ATG6), one of the proteins required for initiation of the formation of autophagic vacuoles, caused reduction in autophagic vacuoles (MDC staining by confocal microscopy) in OCI-AML-3 and REH cells without affecting apoptosis induction (Annexin V by flow cytometry). Pharmacologic inhibition of late autophagy by Bafilomycin (10nM for 2 hours) reduced MDC staining in OCI-AML-3 cells treated with Nutlin-3a for 48 hrs (32% without and 9% with Bafilomycin) while having limited inhibition of apoptosis (Annexin V positive 42% without and 33% with Bafilomycin). Conclusion: Nutlin-3a induces autophagy in leukemia cells by a p53 dependent manner. We also demonstrate that autophagy could go hand-in-hand with apoptosis and in a fraction of cells both processes may occur concomitantly. Inhibition of autophagy does not necessarily enhance apoptosis. Disclosures: Andreeff: Roche: Research Funding. Borthakur:ASCO: Research Funding.

The Novel Sulfonamidebenzamide ML291 Activates Apoptotic UPR Signaling in Pediatric Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3523-3523
Author(s):  
Danielle Garshott ◽  
Nicole Melong ◽  
Tania T. Sarker ◽  
Yue Xi ◽  
Amy Brownell ◽  
...  
Keyword(s):  
Cell Lines ◽  
Leukemia Cell ◽  
Leukemic Cell ◽  
K562 Cells ◽  
Leukemia Cells ◽  
Wild Type ◽  
Leukemia Cell Lines ◽  
Proliferation Assays

Abstract Background: Acute leukemias are the most common cancers in childhood. Despite multi-agent chemotherapy protocols and the introduction of novel molecularly targeted therapies which have resulted in improved survival over the last few decades, relapsed acute lymphoblastic leukemia remains the second most common pediatric cancer diagnosis. In addition, morbidities from current chemotherapy regimens are unacceptably high. Abundant evidence point to a major role for mediators of the unfolded protein response (UPR) in normal and leukemic white blood cell biology. We have demonstrated that activation of the UPR is a productive approach to inhibit the proliferation of solid tumor cell lines in vitro and to reducing xenograft burden in vivo. The UPR consists of genetically distinct mechanisms that serve to clear misfolded proteins from the endoplasmic reticulum (ER) and enhance protein folding, or induce apoptosis if the initiating stress is prolonged or robust. ML291 is a novel UPR-inducing sulfonamidebenzamide, identified through cell-based high throughput screening and iterative SAR-guided chemical synthesis, that overwhelms the adaptive capacity of the UPR and induces apoptosis in a variety of solid cancer models. Objective: To determine the ability of ML291 to activate the UPR and induce apoptosis in a panel of leukemia cell lines, and to use CHOP-null K562 cells to elucidate the relative contribution of the UPR. We hypothesized that ML291 might activate the PERK/eIF2a/CHOP (apoptotic) arm of the UPR and reduce leukemic cell burden in vitro and in vivo. Methods: MTT and luciferase-based proliferation assays, flow cytometry and RT-qPCR were used to evaluate cell growth, UPR activation and apoptosis in a panel of leukemia cell lines that included AML, ALL and CML in cells exposed to ML291. CRISPR-Cas9 genome editing was used to delete CHOP in K562 (human myeloid leukemia) cells. Deletion was validated by immunoblot analysis and these cells were subjected to the same proliferation and gene analyses described above. The in vivo response to ML291 therapy was evaluated in an established zebrafish xenograft assay (Corkery et al. BJH 2011) in which embryos were xenotransplanted with wild type or CHOP knockdown K562 cells and embryos bathed in ML291. Results: Immunoblot and RT-qPCR analysis revealed an accumulation of proteins and increased gene expression for downstream UPR genes, including CHOP, GRP78/BiP, GADD34 and XBP1 in leukemia cells following ML291 treatment, indicating the activation of the UPR. Increased expression of the apoptotic genes, NOXA, PUMA and DR5 was also observed post-treatment with ML291; and dose response proliferation assays performed after 24 hours revealed IC50 concentrations of 1 - 30µM across cell lines. CHOP deleted K562 cells were protected from cell death when cultured with increasing concentrations of ML291, and were significantly less able to translocate phosphatidylserine across the cell membrane and activate the caspase cascade. When zebrafish embryos xenotransplanted with K562-wild type or -CHOP-null cells were bathed in water containing 5mM ML291 for three days, there was a significant reduction in leukemia cell burden exclusively in theK562 wild type xenografts. Conclusion: Collectively these data indicate that intact PERK/eIF2a/CHOP signaling is required for efficient leukemic cell apoptosis in response to ML291 in vitro and in vivo, and support the hypothesis that small molecule enforcement of the UPR might be a productive therapeutic approach in leukemia. Disclosures No relevant conflicts of interest to declare.

scholarly journals Developing a Novel Indolocarbazole as Histone Deacetylases Inhibitor against Leukemia Cell Lines

2015 ◽  
Vol 2015 ◽  
pp. 1-9 ◽  
Author(s):  
Wenjing Wang ◽  
Maomin Lv ◽  
Xiong Zhao ◽  
Jingang Zhang
Keyword(s):  
Cell Lines ◽  
Histone Deacetylases ◽  
Leukemia Cell ◽  
Growth Effect ◽  
Human Leukemia ◽  
Dependent Manner ◽  
High Concentration ◽  
Concentration Dependent Manner ◽  
Histone Deacetylase 1 ◽  
Leukemia Cell Lines

A novel indolocarbazole (named as ZW2-1) possessing HDAC inhibition activity was synthesized and evaluated against human leukemia cell lines HL-60 and NB4. ZW2-1 performed anti-population growth effect which was in a concentration-dependent manner (2–12 μM) by inducing both apoptosis and autophagy in cells. The compound also caused differentiation of HL-60 and NB4 cells as shown by increasing expression of CD11b, CD14, and CD38 at moderate concentration (4 μM). At relatively high concentration (8 μM), ZW2-1 significantly decreased intracellular histone deacetylase 1 level which was also observed. All the results indicated that ZW2-1 could be a novel antileukemia lead capable of simultaneously inducing apoptosis, autophagy, and differentiation.

Laboratory characterization of leukemic cell procoagulants

2017 ◽  
Vol 55 (8) ◽  
pp. 1215-1223 ◽  
Author(s):  
Renáta Hudák ◽  
Ildikó Beke Debreceni ◽  
Ivett Deák ◽  
Gabriella Gál Szabó ◽  
Zsuzsanna Hevessy ◽  
...  
Keyword(s):  
Cell Lines ◽  
Thrombin Generation ◽  
Leukemia Cell ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Monocytic Leukemia ◽  
Time To Peak ◽  
Leukemia Cell Lines ◽  
Normal Human ◽  
The Mean

Abstract Background: In acute myeloid leukemias, there is an increased chance to develop thrombotic disorders. We hypothesized that in addition to leukemic promyelocytes, monocytic leukemia cells may also have a higher procoagulant activity. Methods: Fibrin formation was assessed by a one-stage clotting assay using a magnetic coagulometer. The thrombin generation test (TGT) of magnetically isolated normal human monocytes, intact leukemic cells and their isolated microparticles was performed by a fluorimetric assay. Phosphatidylserine (PS) expression of leukemic cells and microparticle number determinations were carried out by flow cytometry. Results: All cell lines displayed a significant procoagulant potential compared to isolated normal human monocytes. In the TGT test, the mean of lagtime and the time to peak parameters were significantly shorter in leukemic cells (3.9–4.7 and 9.9–10.3 min) compared to monocytes (14.9 and 26.5 min). The mean of peak thrombin in various monocytic leukemia cell lines was 112.1–132.9 nM vs. 75.1 nM in monocytes; however, no significant difference was observed in the ETP parameter. Factor VII-deficient plasma abolished all procoagulant activity, whereas factor XII-deficient plasma did not affect the speed of fibrin formation and thrombin generation but modulated the amount of thrombin. Factor XI-deficient plasma affected the time to peak values in one leukemic cell line and also attenuated peak thrombin. Leukemia cell-derived microparticles from all three cell lines exerted a procoagulant effect by significantly shortening the lagtime in TGT; there was a nonsignificant difference in case of ETP parameter. Conclusions: All investigated monocytic leukemia cell lines exhibited significant thrombin generation. This phenomenon was achieved by the procoagulants on the surface of leukemic cells as well as by their microparticles.

scholarly journals The Ubiquitin Ligase DTX2 Promotes Nuclear Export of RUNX1 and Inhibits RUNX1-Dependent Leukemogenesis

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1252-1252
Author(s):  
Taishi Yonezawa ◽  
Hirotaka Takahasi ◽  
Tatsuya Sawasaki ◽  
Toshio Kitamura ◽  
Susumu Goyama
Keyword(s):  
Cell Lines ◽  
Nuclear Export ◽  
Ectopic Expression ◽  
Leukemia Cell ◽  
Ring Finger ◽  
K562 Cells ◽  
Cytoplasmic Localization ◽  
E3 Ligases ◽  
Leukemia Cell Lines ◽  
Cord Blood Cells

Transcription factor RUNX1 plays important roles in hematopoiesis and leukemogenesis. As a master regulator for hematopoiesis, RUNX1function is tightly controlled through posttranslational modifications, including ubiquitination. We and others have identified multiple E3 ubiquitin ligases that promote RUNX1 ubiquitination, such as STUB1, RNF38 and APC complex. However, mechanisms for ubiquitination-mediated regulation of RUNX1 function, especially the proteasome-independent roles of ubiquitination, have not been fully understood. We previously established a high-throughput AlphaScreen assayto measure the interaction between RUNX1 and E3 ubiquitin ligases.In this assay, biotinylated full-length RUNX1 was captured by streptavidin-coated donor beads and FLAG-tagged E3 ligases were bound by protein A-conjugated acceptor beads. The binding of RUNX1 to each E3 ligase brings the donor and acceptor beads in close proximity, enabling energy transfer between them, resulting in a chemiluminescence signal. This cell-free AlphaScreen assay allows for the efficient detection of RUNX1-interacting E3 ligases with high sensitivity, and have revealed several E3 ligases, including DTX2, that interact with RUNX1. DTX2 is a mammalian homolog of Drosophila deltex and functions as a RING-finger E3 ubiquitin ligase. Interestingly, a RUNX1 fusion gene connected to reversed DTX2 was identified in acute myeloid leukemia with t(7;21). Given that the reversed sequence of DTX2could inhibit activities of wild-type DTX2, the presence of RUNX1-DTX2 (reversed-sequence) chimera in AML indicates the functional association between RUNX1 and DTX2 in hematopoietic cells. To test this possibility, we first examined whether DTX2 physically interacts with RUNX1 to promote its ubiquitination. Immunoprecipitation assays revealed that DTX2 in fact interacted with RUNX1 in 293T cells. We also found that DTX2, but not a DTX2 mutant lacking Ring finger domain (DTX2-DR), induced ubiquitination of RUNX1 in both nucleus and cytoplasm. However, DTX2-induced RUNX1 ubiquitination did not result in degradation of RUNX1 protein, suggesting that DTX2 modifies RUNX1 function mainly through a proteasome-independent mechanism. We therefore examined if DTX2 alters subcellular localization of RUNX1 using immunofluorescence analysis. Strikingly, ectopic expression of DTX2 altered RUNX1 distribution from nuclear towards cytoplasm in 293T cells (Figure 1). Furthermore, we observed similar cytoplasmic localization of endogenous RUNX1 in DTX2-transduced K562 cells. We then assessed whether DTX2 is involved in RUNX1-mediated transcriptional regulation using the MCSFR luciferase reporter, and found that RUNX1-induced reporter activation was inhibited by ectopic expression of DTX2. Taken together, these data suggest that DTX2 promotes nuclear export of RUNX1, thereby inhibits its transcriptional activity. Next, we assessed the effect of DTX2 on RUNX1 function in primary human cord blood cells. In agreement with previous reports, RUNX1 overexpression promoted myeloid differentiation and downregulated a stem cell marker CD34 in cord blood cells. This RUNX1-induced CD34 downregulation was attenuated by DTX2 coexpression, suggesting the inhibitory effect of DTX2 on RUNX1 activity. We then examined the effect of DTX2 expression in several leukemia cell lines: Jurkat, TF-1, Kasumi-1, SKNO-1 and K562 cells. Previous studies have shown that Jurkat, TF-1, Kasumi-1 and SKNO-1 cells are dependent on RUNX1 activity for their growth. In contrast, K562 cells were shown to be relatively resistant to RUNX1 depletion. Consistent with our earlier results, expression of DTX2 promoted cytoplasmic localization of RUNX1 in all these leukemia cell lines. Functionally, DTX2 overexpression inhibited the growth of RUNX1-dependent leukemia cell lines (Jurkat, TF-1, Kasumi-1, and SKNO-1) by inducing apoptosis and cell-cycle arrest, while it showed only modest effect in K562 cells. Thus, these results indicate again that DTX2 inhibit RUNX1 function by promoting its cytoplasmic localization in hematopoietic and leukemia cells. In summary, we identified DTX2 as an E3 ligase to promote RUNX1 ubiquitination. DTX2 does not induce RUNX1 degradation, but instead promotes nuclear export of RUNX1 to inhibit its function. Activation of DTX2 could be a therapeutic approach to treat RUNX1-dependent leukemias. Disclosures No relevant conflicts of interest to declare.

scholarly journals Effect of tamoxifen on cell lines displaying the multidrug-resistant phenotype [see comments]

Blood ◽  
1991 ◽  
Vol 77 (4) ◽  
pp. 818-825
Author(s):  
E Berman ◽  
M Adams ◽  
R Duigou-Osterndorf ◽  
L Godfrey ◽  
B Clarkson ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Leukemia Cell ◽  
Multidrug Resistant ◽  
Leukemia Cell Line ◽  
Parent Cell ◽  
Continuous Exposure ◽  
Dependent Manner ◽  
Uptake Pattern ◽  
Dose Dependent

We examined the effect of tamoxifen (Tmx), verapamil, and daunorubicin (DNR) in two cell lines that displayed the multidrug-resistant (MDR) phenotype and used laser flow cytometry to quantitate intracellular DNR content. In the vinblastine-resistant human lymphoblastic lymphoma cell line CEM-VBL, simultaneous incubation of DNR with Tmx 10 mumol/L or Tmx 50 mumol/L increased intracellular DNR fluorescence in a dose-dependent manner and demonstrated an uptake pattern similar to that seen with DNR and verapamil. Similar results were obtained in the vincristine- resistant human myeloid leukemia cell line HL-60/RV+. Cellular retention of DNR was also measured in both cell lines and results suggested that continuous exposure of the cells to Tmx resulted in higher intracellular DNR content compared with cells resuspended in fresh medium. No effect of Tmx or verapamil was observed in the drug- sensitive parent cell lines CEM or HL-60. Clonogenic experiments were then performed to determine whether Tmx was itself inhibitory to cell growth or whether Tmx potentiated DNR cytotoxicity. Tmx 10 mumol/L did not significantly inhibit either CEM-VBL or HL-60/RV+ cells after a 3- hour exposure followed by culture in methylcellulose. Tmx 50 mumol/L was significantly more inhibitory in both cell lines. However, cells that had been incubated with DNR and Tmx 10 mumol/L demonstrated a marked increment in growth inhibition compared with cells that had been incubated with DNR alone or Tmx 10 mumol/L alone. Based on the data presented here, we suggest that clinical testing of Tmx and DNR be pursued in the setting where MDR may play a role.

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