scholarly journals Response of newly established mouse myeloid leukemic cell lines to MC3T3-G2/PA6 preadipocytes and hematopoietic factors

Blood ◽  
1991 ◽  
Vol 77 (1) ◽  
pp. 49-54 ◽  
Author(s):  
H Kodama ◽  
M Iizuka ◽  
T Tomiyama ◽  
K Yoshida ◽  
M Seki ◽  
...  
Keyword(s):  
Cell Growth ◽  
Cell Lines ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Hematopoietic Stem ◽  
Leukemic Cell Lines ◽  
Hematopoietic Factors ◽  
In Vitro Cell Lines

Abstract Some mouse myeloid leukemias induced by X-irradiation and serially transplanted into syngenic mice do not proliferate in vitro even in the presence of hematopoietic factors. To examine whether such leukemic cells can proliferate in response to stromal cells, we cocultured them with MC3T3-G2/PA6 (PA6) preadipocytes, cells that can support the growth of hematopoietic stem cells. All leukemias developed into in vitro cell lines, showing a dependence on contact with the PA6 cells. Two cell lines responded to none of the known hematopoietic factors including interleukin-3 (IL-3), IL-4, IL-5, IL-6, GM-CSF, G-CSF, M-CSF, and Epo. These results demonstrate that the mechanism of the action of PA6 cells is different from that of any of the known hematopoietic factors, and that, because these two leukemic cell lines retained the ability to grow in vivo, responsiveness to the known hematopoietic factors is not essential for the leukemic cell growth in vivo. Furthermore, all leukemic cell lines could respond also to the preadipocytes fixed with formalin, paraformaldehyde, or glutaraldehyde, suggesting that some molecule(s) associated with the surface of PA6 cells or with extracellular matrix secreted by the preadipocytes is responsible for the leukemic cell growth.

scholarly journals Response of newly established mouse myeloid leukemic cell lines to MC3T3-G2/PA6 preadipocytes and hematopoietic factors

Blood ◽  
1991 ◽  
Vol 77 (1) ◽  
pp. 49-54
Author(s):  
H Kodama ◽  
M Iizuka ◽  
T Tomiyama ◽  
K Yoshida ◽  
M Seki ◽  
...  
Keyword(s):  
Cell Growth ◽  
Cell Lines ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Hematopoietic Stem ◽  
Leukemic Cell Lines ◽  
Hematopoietic Factors ◽  
In Vitro Cell Lines

Some mouse myeloid leukemias induced by X-irradiation and serially transplanted into syngenic mice do not proliferate in vitro even in the presence of hematopoietic factors. To examine whether such leukemic cells can proliferate in response to stromal cells, we cocultured them with MC3T3-G2/PA6 (PA6) preadipocytes, cells that can support the growth of hematopoietic stem cells. All leukemias developed into in vitro cell lines, showing a dependence on contact with the PA6 cells. Two cell lines responded to none of the known hematopoietic factors including interleukin-3 (IL-3), IL-4, IL-5, IL-6, GM-CSF, G-CSF, M-CSF, and Epo. These results demonstrate that the mechanism of the action of PA6 cells is different from that of any of the known hematopoietic factors, and that, because these two leukemic cell lines retained the ability to grow in vivo, responsiveness to the known hematopoietic factors is not essential for the leukemic cell growth in vivo. Furthermore, all leukemic cell lines could respond also to the preadipocytes fixed with formalin, paraformaldehyde, or glutaraldehyde, suggesting that some molecule(s) associated with the surface of PA6 cells or with extracellular matrix secreted by the preadipocytes is responsible for the leukemic cell growth.

scholarly journals PARP Inhibition Sensitize BCR-ABL1 Positive Cel

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3367-3367 ◽  
Author(s):  
Haruka Hiroki ◽  
Masatoshi Takagi ◽  
Yuko Ishi ◽  
Jinhua Piao ◽  
Tomohiro Morio
Keyword(s):  
Cell Death ◽  
Cell Lines ◽  
Parp Inhibitor ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Parp Inhibition ◽  
Akt Pathway ◽  
Leukemic Cell Lines

Introduction: BCR-ABL1 play a key role in the development of chronic myelogenous leukemia and a part of Ph1 positive acute lymphoblastic leukemia (ALL). BCR-ABL1 functions as a tyrosine kinase. Whereas, BCR-ABL1 induces genomic instability by downregulation of BRCA1. An innate error of BRCA1, a molecule involved in the homologous recombination repair pathway, causes hereditary breast and ovarian cancer. PARP inhibitor (PARPi) induces synthetic lethality in BRCA defective cell. Therefore, PARP inhibitor is expected to induce efficient cell death with BCR-ABL1 positive cell. In addition, in some previous reports, reduction of PARP1 activity leads to the upregulation of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway and BCR-ABL1 tyrosine kinase activates PI3K/AKT pathway. These findings suggest activation of the PI3K/AKT pathway leading to PARPi resistance in BCR-ABL1 positive leukemic cells. Here, we demonstrate that PARP inhibition attenuates BCR-ABL1 mediated leukemogenesis and aberration of factors associated with PARP inhibitor resistance induces cell death to fully transformed leukemic cells. Method: Bone marrow-derived mononuclear cells (MNC) from wild type mice and BCR-ABL1 transgenic (Tg) mice were exposed to PARPi in vivo, and cell death was analyzed Annexin-V positivity. PARPi sensitivity to BCR-ABL1 expressed cell was also investigated in vivo bone marrow transplantation model using mouse hematopoietic stem cell (HCS) infected with BCR-ABL1 expressing retrovirus. To evaluate more precisely the results obtained in vitro and in vivo transplantation model, the genetical approach was also performed. The Parp1 knockout (KO) mice were crossed with BCR-ABL1 Tg mice. Then, Leukemia development and subsequent mouse death were observed. In vitro, HR activity was examined using DR-GFP assay. Genomic instability was investigated using the breakage-fusion-bridge (BFB) generation.Maintenance of HSC as a progenitor of the leukemic cell was analyzed by repopulation activity using colony assay. The growth-inhibitory effect was assessed using BCR-ABL positive cell lines with PARPi and PI3K inhibitor. Results: BCR-ABL1 Tg mice derived MNC showed more hypersensitivity to PARPi. Mouse HCS was infected with BCR-ABL1 expressing retrovirus and transplanted lethally Olaparib or vehicle was administrated intraperitoneal injection one day after transplantation. BCR-ABL1 mediated leukemic death was observed 1 month after transplantation in sham-treated mouse, whereas, Olaparib treated mouse did not develop BCR-ABL1 mediated leukemia. Parp1 KO BCR-ABL1 Tg mice attenuated leukemia development and extended their survival compared with BCR-ABL1 Tg mice. In vitro experiment revealed HR activity was down-regulated by BCR-ABL1 expression in DR-GFP assay. The number of BFB generation was increased in BCR-ABL1 Tg with Parp1 KO background. The colony-forming activity of BCR-ABL1 positive HSC was totally abolished by PARP inhibition after 3 times serial replating, whereas sham-treated HSC retained repopulation activity. However, the effect of PARPi on BCR-ABL positive leukemic cell lines was controversial. Therefore, leukemic cell lines were treated with the PARPi and inhibitors toward the molecules associated with PARPi resistance. As a result, a combination of PARPi with PI3K inhibitor effectively induce cell death in PARPi resistant BCR-ABL1 positive leukemic cell lines. Conclusion and discussion: Tyrosine kinase inhibitor (TKI) is the gold standard of the therapeutic option of BCR-ABL1 positive leukemia. However, TKI monotherapy is not sufficient for complete eradication of leukemic cells. It is highly expected that molecules effectively induce cell death to leukemic cells combined with TKI. PARPi would be one of these candidates. However, PARPi could not induces efficient death in all of the cancer cells that carry the mutation of molecules associated with the HR defect. Comprehensive genetic analysis to reveal PARPi resistance is important for HRR defective cancer cells. Combination therapy of PARPi and inhibitorstoward the molecules associated with PARPi resistance would be a good therapeutic option for Ph1 positive leukemia. Disclosures No relevant conflicts of interest to declare.

Differential expression of a novel proline-rich homeobox gene (Prh) in human hematolymphopoietic cells

Blood ◽  
1995 ◽  
Vol 85 (5) ◽  
pp. 1237-1245 ◽  
Author(s):  
G Manfioletti ◽  
V Gattei ◽  
E Buratti ◽  
A Rustighi ◽  
A De Iuliis ◽  
...  
Keyword(s):  
Cell Lines ◽  
Constitutive Expression ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Maturation Stage ◽  
Lymphoid Tissues ◽  
Specific Cell ◽  
B Cell Differentiation ◽  
Leukemic Cell Lines

Proline-rich homeobox (Prh) is a novel human homeobox-containing gene recently isolated from the CD34+ cell line KG-1A, and whose expression appears mainly restricted to hematopoietic tissues. To define the pattern of Prh expression within the human hematopoietic system, we have analyzed its constitutive expression in purified cells obtained from normal hematopoietic tissues, its levels of transcription in a number of leukemia/lymphoma cell lines representing different lineages and stages of hematolymphopoietic differentiation, and its regulation during in vitro maturation of human leukemic cell lines. Prh transcripts were not detected in leukemic cells of T-lymphoid lineage, irrespective of their maturation stage, and in resting or activated normal T cells from peripheral blood and lymphoid tissues. In contrast, high levels of Prh expression were shown in cells representing early stages of B lymphoid maturation, being maintained up to the level of circulating and tissue mature B cells. Terminal B-cell differentiation appeared to be conversely associated with the deactivation of the gene, since preplasmacytic and plasmocytoma cell lines were found not to express Prh mRNA. Prh transcripts were also shown in human cell lines of early myelomonocytic, erythromegakaryocytic, and preosteoclast phenotypes. Prh expression was lost upon in vitro differentiation of leukemic cell lines into mature monocyte-macrophages and megakaryocytes, whereas it was maintained or upregulated after induction of maturation to granulocytes and osteoclasts. Accordingly, circulating normal monocytes did not display Prh mRNA, which was conversely detected at high levels in purified normal granulocytes. Our data, which show that the acquisition of the differentiated phenotype is associated to Prh downregulation in certain hematopoietic cells but not in others, also suggest that a dysregulated expression of this gene might contribute to the process of leukemogenesis within specific cell lineages.

scholarly journals Akacid Medical Formulation Induces Apoptosis in Myeloid and Lymphatic Leukemic Cell Lines In Vitro and In Vivo

PLoS ONE ◽  
2015 ◽  
Vol 10 (2) ◽  
pp. e0117806 ◽  
Author(s):  
Hannes Neuwirt ◽  
Elisabeth Wabnig ◽  
Clemens Feistritzer ◽  
Iris E. Eder ◽  
Christina Salvador ◽  
...  
Keyword(s):  
Cell Lines ◽  
Leukemic Cell ◽  
Leukemic Cell Lines

Pioglitazone Inhibits the Growth of Human Leukemic Cell Lines and Primary Leukemic Cells in Vitro.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4493-4493 ◽  
Author(s):  
Yoshihiro Hatta ◽  
Minoru Saiki ◽  
Yuko Enomoto ◽  
Shin Aizawa ◽  
Umihiko Sawada ◽  
...  
Keyword(s):  
Cell Lines ◽  
Colony Formation ◽  
Mononuclear Cells ◽  
Hematopoietic Cells ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Peroxisome Proliferator ◽  
Leukemic Cell Lines ◽  
Ppar Γ

Abstract Troglitazone and pioglitazone are one of thiazolidinediones that are high affinity ligand for the nuclear receptor called peroxisome proliferator-activated receptor gamma (PPAR-γ). Troglitazone is a potent inhibitor of clonogenic growth of acute myeloid leukemia cells when combined with a retinoid. However, the effect of pioglitazone to neoplastic cells and normal hematopoietic cells has not been studied yet. Adult T-cell leukemia (ATL), prevalent in western Japan, is a highly aggressive malignancy of mature T lymphocyte. Therefore, we studied antitumor effect of pioglitazone against leukemic cells including ATL as well as normal hematopoietic cells. With 300 μM of pioglitazone, colony formation of ATL cell lines (MT1, MT2, F6T, OKM3T, and Su9T01) was completely inhibited. Colony formation of HUT102, another ATL cell line, was 12 % compared to untreated control. Clonogenic cells of other leukemic cell lines (K562, HL60, U937, HEL, CEM, and NALM1) was also inhibited to 0–30% of control. Colony formation of primary leukemic cells from 5 AML patients was decreased to 15 %. However, normal hematopoietic cells were weakly inhibited with 300 μM pioglitazone; 77 % of CFU-GM, 70 % of CFU-E, and 33 % of BFU-E survived. Cell cycle analysis showed that pioglitazone decreased the ratio of G2/M phase in HL60 cells, suggesting the inhibition of cell division. By Western blotting, PPAR-γ protein level was similar in all leukemic cells and normal bone marrow mononuclear cells. Taken together, pioglitazone effectively eliminate leukemic cells and could be used as an antitumor agent in vivo.

Expression and Function of the CXCR7 Chemokine Receptor in Leukemias

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2423-2423
Author(s):  
Sergej Konoplev ◽  
Hongbo Lu ◽  
Michael A Fiegl ◽  
Zhihong Zeng ◽  
Wenjing Chen ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Human Leukemia ◽  
Rt Pcr ◽  
Hematopoietic Stem ◽  
Leukemic Cell Lines ◽  
And Function

Abstract Background: Bone marrow produced stromal-derived factor-1a (SDF-1a) is a key chemokine involved in chemotaxis, homing, mobilization, and expansion of hematopoietic stem and progenitor cells. While the majority of well-defined functions of SDF-1a are mediated via its receptor CXCR4, recent studies have characterized CXCR7 as an alternative receptor capable of binding SDF-1a. Although the functions of CXCR7 are still incompletely understood, the receptor was reported to promote migration and adhesion in certain cell types and function as a pro-survival factor in breast cancer cells. CXCR7 expression and function in human leukemia cells has not been characterized. In this study, we examined CXCR7 expression in leukemia cell lines and primary samples from patients with acute lymphoblastic leukemia (ALL) and utilized a small molecule inhibitor of CXCR7 to probe CXCR7’s function. Materials and methods: CXCR4 and CXCR7 expression was determined by flow cytometry, real-time PCR (RT-PCR) and immunocytochemistry (ICC) in leukemic cell lines including AML (OCI-AML2, OCI-AML3, HL60, U937 NB4, Molm13), ALL (REH, Raji, RS4; 11, Nalm6, Molt4) and CML (KBM5, K562) cells. In primary ALL patient samples, CD34+CD19+ gating was applied to detect CXCR7 expression on pre-B leukemic cells by flow cytometry. The migration of leukemic cells towards SDF-1a was studied using a transwell system. CXCR4 inhibitor AMD3100 was purchased from Sigma, and CXCR7 inhibitor CCX-733 was provided by ChemoCentryx Inc., Mountain View, CA. Results: CXCR4 was found to be ubiquitously expressed on the cell surface of all leukemic cell lines tested. CXCR7 mRNA and protein expression was detectable only in Burkitt lymphoma Raji cells, as analyzed by flow cytometry (clone 11G8, R&D systems), RT-PCR and ICC. Curiously, CXCR7 expression was significantly induced in MOLM13 cells under hypoxic (6% O2) conditions (p=0.01). Low levels of surface CXCR7 were found in 8 of the 9 primary ALL samples by flow cytometry. To determine the respective roles of CXCR4 and CXCR7 in migration of leukemic cells, we utilized CXCR4 inhibitor AMD3100 and CXCR7 inhibitor CCR733 in Raji (CXCR7 positive) and RS4;11 (CXCR7 negative) cells. AMD3100 at 25μM significantly inhibited SDF-1a induced migration (from 38.5% to 12%); CCR733 at 10μM also inhibited SDF-1a induced migration (from 38.5% to 24%) and the combination of AMD3100 and CCR733 resulted in 81% inhibition of migration (from 38.5% to 7.2%). AMD3100 blocked SDF-1a induced migration of CXCR4+CXCR7− RS4;11 cells (from 36.5% to 15.8%), while CCR733 had no effect (36.5% and 39.2%). In conclusion, these studies demonstrate functional expression of the SDF-1 receptor CXCR-7 on Raji and primary ALL cells and suggest that CXCR7 plays an active role in the migration of leukemic cells. CXCR-7 may serve as an alternative receptor to CXCR4. Studies addressing the role of CXCR7 in adhesion, SDF-1a-mediated signaling and survival of leukemic cells are in progress.

Chemoprevention Against Hepatocellular Carcinoma of Cornus officinalis in vitro

2004 ◽  
Vol 32 (05) ◽  
pp. 717-725 ◽  
Author(s):  
Jung-San Chang ◽  
Lien-Chai Chiang ◽  
Fen-Fang Hsu ◽  
Chun-Ching Lin
Keyword(s):  
Hepatocellular Carcinoma ◽  
Cell Lines ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Xtt Assay ◽  
Leukemic Cell Lines ◽  
Cornus Officinalis ◽  
Anti Oxidant ◽  
Dose Dependent

The water extracts of Cornus officinalis Sieb. et Zuce against hepatocellular carcinoma (HCC) was studied for its chemopreventive potential. Three HCC cell lines (HepG2, SK-Hep1 and PLC/PRF/5) and three leukemic cell lines (U937, K562 and Raji) were tested with XTT assay. Extracts of C. officinalis inhibited all these HCC cells and leukemic cells at a concentration of 100 μg/ml (P<0.05) and was dose-dependent (P<0.0001). P53 (P<0.0001) and Ras (P=0.001) significantly affected its activity against HCC. Extracts of C. officinalis also possessed the anti-oxidant activity through free radicals scavenging activity at a concentration of 50 μg/ml (P<0.05). In summary, our experiment implied that C. officinalis might be a candidate for chemopreventive agent against HCC through the antioxidant and anti-neoplastic effects.

Sulforaphane Switches Cyclin E Isoform Expression and Blocks Leukemic Cell Cycling.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4405-4405
Author(s):  
Laura M. Gorham ◽  
Gigi Frye ◽  
Michelle Miranda ◽  
Richard A. Steinman
Keyword(s):  
Molecular Weight ◽  
Cyclin E ◽  
Leukemic Cell ◽  
Full Length ◽  
Leukemic Cells ◽  
Pharmacokinetic Studies ◽  
Leukemic Cell Lines ◽  
Myeloid Leukemic Cell

Abstract Sulforaphane (SFN), an isothiocyanate derived from cruciferous vegetables, has been shown to inhibit the growth of prostate cancer cells in vitro and in vivo. We were interested in exploring potential antileukemic effects of SFN. The viability of multiple myeloid leukemic cell lines was decreased by 25uM SFN. Pharmacokinetic studies reported in rats suggest that this serum concentration can be achieved through oral dosing. Lower SFN concentrations (1–5 uM) inhibited leukemic cell growth without affecting cell viability. Synchronized HL-60 cells exposed to 25uM SFN were blocked at the G1/S phase transition. Kinetic analysis of cell cycle proteins demonstrated that the G1/S block arose from downmodulation of cyclins D3 and E rather than upregulation of cdk-inhibitors. Interestingly, we found that HL-60 cells expressed a low molecular weight (LMW, 36 kD) variant of cyclin E rather than (50 kD) full-length cyclin E. Treatment with SFN for as little as 2 hours caused a decrease in expression of the LMW cyclin E and induced the expression of a higher molecular weight (~50 kD) cyclin E isoform. Because LMW cyclin E has been associated with increased cdk2 activity and p27 resistance compared to full-length cyclin E, we postulate that SFN-mediated cyclin E isoform-switching contributed to growth inhibition of these leukemic cells. The signaling pathway through which SFN altered cyclin E expression appeared to be distinct from MEK/ERK and JNK pathways that have been implicated in the apoptotic effects of SFN. Given that cyclin E overexpression and, particularly, LMW cyclin E expression are correlated with poor prognosis in multiple cancers, the mechanism through which SFN decreases LMW cyclin E expression in these leukemic cells could have therapeutic significance.

Metabolic Capability to Induce the Pasteur Effect Mediates Sensitivity of Human Leukemic Cells to Metformin

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2601-2601
Author(s):  
Sarah Scotland ◽  
Estelle Saland ◽  
Lindsay Peyriga ◽  
Rémi Peyraud ◽  
Elizabeth Micklow ◽  
...  
Keyword(s):  
Cell Lines ◽  
Research Funding ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Pasteur Effect ◽  
Cytotoxic Response ◽  
Leukemic Cell Lines

Abstract Abstract 2601 An emerging hallmark of cancer cells is the reprogramming of intermediary and energy metabolism these cells undergo. Several epidemiological studies have shown that metformin, widely used to treat patients with type 2 diabetes, may reduce their risk of cancer. Despite several reports of anti-neoplastic activity of metformin, the mechanisms responsible for this activity have not been fully elucidated in cancer or leukemic cells. We hypothesized that metformin elicits a metabolic reprogramming driven by alterations in mitochondrial function and signaling, which induces apoptosis in leukemic cells, and that metabolic flexibility determines the variation(s) of the cytotoxic response to metformin among different leukemic cell lines. We first demonstrated that metformin markedly decreased oxygen consumption of six leukemic cell lines in a concentration-dependent manner. We also observed that the cytotoxic effect of metformin varies between cell lines reflecting their energetic capacity to compensate for the mitochondrial inhibition induced by metformin (eg. to induce the Pasteur effect). Importantly, metformin-insensitive leukemic cells did not exhibit a Pasteur effect in response to metformin. All leukemic cells exhibited high basal conversion of glucose to lactate (eg. aerobic glycolysis) and specific expression of key metabolic genes as compared to normal mononuclear cells. Despite dependence on glucose catabolism, metformin sensitivity was associated with relative resistance to glucose starvation. Metformin effects in drug-resistant cells were potentiated by the addition of a glycolytic inhibitor, but not by inhibitors of the pentose phosphate pathway or glutaminolysis. Leukemic cells with broad metabolic capacities to utilize other energetic substrates in response to diverse nutrient starvation showed insensitivity to metformin. Metformin induced a significant decrease in metabolites of the upper segment of glycolysis and the oxidative branch of the pentose phosphate pathway as well as a clear increase of PRPP and IMP biosynthesis. Energy charge, the nucleotide phosphate pool and lactate/glucose ratio remained stable after metformin treatment. Furthermore, our results showed that basal glucose uptake/consumption and the activity of the lower segment of the glycolytic pathway are key determinants of a cytotoxic response to metformin. In addition, high glutathione, malate, IMP and orotate content were observed in metformin-insensitive leukemic cells. Moreover, the cytotoxic effect of metformin was independent of AMPK/LKB1 status of the leukemic cells while p53 expression abrogated this effect. The presence of wild-type p53 appears to partially protect tumor cells from glucose starvation and metformin cytotoxicity and prevents the induction of the Pasteur effect. Finally, we demonstrated that metformin increased the cytotoxicity of chemotherapy agent, cytarabine, on all leukemic cell lines in vitro and significantly reduced leukemic colony-forming units (CFU-L) from six primary AML patient samples in a concentration-dependent manner. Additional experiments on metabolic and signaling pathways as well as in vivo studies are in progress to better understand the cytotoxic response of metformin in both AML cell lines and primary AML patient specimens that impact the therapeutic potential of metformin in vivo. Disclosures: Carroll: Agios Pharmaceuticals: Research Funding; TetraLogic Pharmaceuticals: Research Funding; Sanofi Aventis Corporation: Research Funding; Glaxo Smith Kline, Inc.: Research Funding.

Targeting Aberrant Self-Renewal of Leukemic Cells with a Novel CBP/p300 Bromodomain Inhibitor

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3750-3750
Author(s):  
Angeliki Thanasopoulou ◽  
Katharina Dumrese ◽  
Sarah Picaud ◽  
Oleg Fedorov ◽  
Stefan Knapp ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Lines ◽  
Colony Formation ◽  
Leukemia Cell ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Human Leukemia ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Leukemic Cell Lines

Abstract The CBP/p300 histone acetyltransferases are key transcriptional regulators of hematopoiesis that have been found to be involved in AML-associated recurrent chromosomal translocations and shown to function as co-activators of leukemogenic fusion oncogenes, suggesting that specific targeting of CBP/p300 may be beneficial for therapy. We characterized the anti-leukemic potential of I-CBP112, a novel chemical inhibitory probe targeting the CBP/p300 bromodomain (BRD). BRDs belong to a diverse family of evolutionary conserved protein-interaction modules recognizing acetylated lysine residues and thereby mediating recruitment of proteins to macromolecular complexes. I-CBP112 represents a new, potent and selective class of BRD inhibitors (oxazepines) binding to recombinant CBP/p300 BRDs with a KD of 151nM and 157nM respectively. Initial characterization by FRAP and BRET assays revealed that I-CBP112 displaced the isolated BRD construct from chromatin but not the full length CBP. I-CBP112 also impaired the interaction of CBP/p300 with p53, resulting in reduced p53-K382 acetylation, reduced p21 expression, and high sensitivity to Doxorubicin-induced DNA damage. We started to explore the effects of the compound on leukemic cells by exposing a series of murine cell lines immortalized by the MLL-CBP fusion and other potent leukemia-associated oncogenes including the MLL-AF9, MLL-ENL, or the NUP98-HOXA9 fusion to increasing doses of I-CBP112. Interestingly, no significant cytotoxicity was observed up to concentrations of 5μM. However, in all cell lines we observed a significant reduced number of colonies formed in methylcellulose, associated with morphological differentiation as observed in Giemsa stained cytospots. Similar to the murine leukemic cell lines we found that I-CBP112 did not cause immediate cytotoxic effects but impaired colony formation and induced cellular differentiation of a series of 18 human leukemic cell lines. Reduced colony formation upon I-CBP112 treatment was also observed of human primary AML blasts but not of CD34+ hematopoietic stem cells from two healthy donors. I-CBP112 effects were studied in more detail in three human leukemia cell lines: SEM (MLL-AF4+), MOLM13 (MLL-AF9+) and Kasumi-1 (AML1-ETO+). Long-term exposure of these cells to I-CBP112 in liquid medium, resulted in a dose-dependent G1 cell cycle arrest, with Kasumi-1 being the most sensitive to the inhibitor, demonstrating further morphological signs of differentiation and apoptotic cell death. Importantly, combination of I-CBP112 with the BET-BRD inhibitor JQ1 or Doxorubicin revealed a clear synergistic effect on cell survival of the AML cell lines except for the combination of I-CBP112 with Doxorubicin on MOLM13. Surprisingly only modest effects of I-CBP112 exposure on the transcriptional programs of SEM, MOLM13 and Kasumi-1 cells were found by microarray expression profiling. Genes found affected were mainly immune response regulators or NFkappaB targets suggesting that attenuation of NFkappaB downstream signals might impair the leukemia initiation capacity reflected by reduced colony formation. Extreme limited dilution assays (ELDA) in methylcellulose, as well as bone marrow transplantations in limiting dilutions using MLL-AF9-transformed murine leukemic blasts revealed that I-CBP112 significantly impaired self-renewal of the leukemic stem cell compartment in vitro and reduced the leukemia-initiating potential in vivo. Taken together, these data demonstrate that selective interference with the CBP/p300 BRD by I-CBP112 has the potential to selectively target leukemic stem cells and opens the way for novel combinatory “BRD inhibitor” therapies for AML and other human cancers. Disclosures No relevant conflicts of interest to declare.

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