scholarly journals Enhancing Functional Platelet Release In Vivo from in Vitro-Grown Megakaryocytes Using the Protein Kinase Inhibitor SU6656

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1029-1029
Author(s):  
Danuta Jadwiga Jarocha ◽  
Karen K Vo ◽  
Randolph B Lyde ◽  
Vincent M Hayes ◽  
Mortimer Poncz
Keyword(s):  
Kinase Inhibitor ◽  
Conflicts Of Interest ◽  
Specific Inhibitor ◽  
Annexin V ◽  
Rock Inhibitor ◽  
Medicine Research ◽  
Nsg Mice ◽  
Platelet Release

Abstract The clinical demand for platelet transfusions is increasing, threatening the ability to obtain sufficient healthy donors to provide these platelets. Advances in regenerative medicine research have opened the possibility of generating sufficient in vitro-grown megakaryocytes and consequent platelets to supply a portion of the clinical platelet transfusion demand. We have shown that infusing megakaryocytes for obtaining released, functional platelets is a viable alternative strategy than trying to release platelets in vitro. However, for both approaches, in vitro-cultured megakaryocytes have lower ploidy and release fewer platelets than likely occurs in vivo by primary cells. SU6656 inhibitor, a Src kinase inhibitor, has been shown to influence ploidization in several megakaryocyte-like line with purported increase in proplatelets release. However, in our hands, other agents - such as the ROCK inhibitor Y27632 - while increasing polyploidization markedly, inhibited platelet release per infused megakaryocyte in vivo. We grew megakaryocytes from CD34+ cells for 12 days with or without SU6656 (2.5 µM) supplementation during the last 4 days. We found that the SU6656 inhibitor only increased the number of CD34+-derived megakaryocytes by ~15% at the end of the 12 day growth, but more markedly increase the percent of large megakaryocytes measured by FSC parameter in flow cytometry evaluation from 28 up to 41% and percent of high granular megakaryocytes from 27 to 45%. These changes were accompanied with a shift in average ploidy from 4.9 to 6.9 (p<0.0003, N=6). Notably, SU6656-treated megakaryocytes released ~4-fold more platelets per infused megakaryocytes in immunocompromized NSG mice than untreated similarly in vitro-grown megakaryocytes. By 24 hrs, there were 6.5-fold platelets from the infused SU6656-treated megakaryocytes than control untreated (p<0.037, N=6). Released platelets from the drug-treated and untreated megakaryocytes had similar levels of percent thiazole orange positivity as an indication that they were young platelets. Importantly, baseline annexin V, CD62p and PAC1 binding prior to agonist exposure were also similarly and increased to the same extent after thrombin (1U/ml) stimulation. Additionally, incorporation into a growing cremaster laser injury-induced thrombus in vivo was similar further indicating retained function by the platelets released from the drug-treated megakaryocytes. A number of strategies such as modifying the level of transcription factors have been proposed to increase the size, ploidy or proplatelets release from in vitro-grown megakaryocytes. In none of these cases have these released platelets in vivo biology been examined and demonstrated to replicate high release number per megakaryocyte and retained functionality. We show that terminal exposure of in vitro-grown megakaryocytes to the non-specific inhibitor SU6656 significantly increases in vivo yield while leaving in vivo half-life and functionality intact. The exact pathway affected by SU6656 that leads to these results is now being pursued. Disclosures No relevant conflicts of interest to declare.

Inhibition Of Erythropoiesis By Iron Overload Is Mediated Through TGFβ Signaling

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2787-2787
Author(s):  
Krishna Gundabolu ◽  
Huiyong Chen ◽  
Huihui Li ◽  
Lohith Shakaladevanapura ◽  
Tushar D. Bhagat ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Iron Overload ◽  
Kinase Inhibitor ◽  
Conflicts Of Interest ◽  
Specific Inhibitor ◽  
Cellular Damage ◽  
Erythroid Precursors ◽  
Tgfβ Receptor

Abstract Several diseases, such as β-thalassemia and myelodysplastic syndrome, are characterized by iron overload and dysfunction erythropoiesis. Multiple retrospective studies suggest that iron overload is associated with increased disease burden and shortened survival in patients. It is general consensus that iron overload has an inhibitory effect on erythroid differentiation, but direct evidence is lacking and the mechanisms underlying this phenomenon are incompletely understood. Excess iron is associated with the generation of reactive oxygen species (ROS) that result in cellular damage. We hypothesized that iron overload leads to the stimulation of myelosuppressive pathways that result in decreased erythropoiesis and resulting cytopenias. We first evaluated the effect of exogenous iron on the growth of primary human CD34+ stem cells and demonstrated that iron overload leads to decreased erythroid colony formation in vitro. Furthermore, in our in vivo experiments, we administered a total of 130 mg iron dextran IP over 20 days to C57BL/6 mice and compared these mice with PBS injected age and gender matched controls (n=5/group). We demonstrate that iron injection results in fewer circulating reticulocytes (200 vs. 310 x 109 cells/L; P=0.001) and hemoglobin (13.8 vs. 15 g/dL; P=0.0004). Using anti-CD44 and TER119 as flow cytometric markers, our data reveals a reduction in bone marrow erythroid burden (total erythroid precursors 9.6 vs. 15.3% in controls; P=0.04) with a disproportionately greater effect on orthochromatophilic erythroblasts (4.4 vs. 6.9%; P=0.02). As expected, bone marrow-derived erythroid precursors from iron loaded mice have more ROS (MFI 1497 vs. 958; P=0.05) as measured by flow cytometry, again with a disproportionately greater effect on orthochromatophilic erythroblasts (MFI 671 vs. 403; P=0.01). No effect on cell cycle was observed. To evaluate the mechanism behind iron induced suppression of erythropoiesis, we conducted a functional screen with variety of cytokine inhibitors. We observed that a specific inhibitor of the TGFβ receptor I kinase led to reversal of iron induced suppression of erythroid colonies from primary CD34+ cells in vitro. The ability of TGFβ kinase inhibitor, LY-215, in reversing iron mediated suppression was confirmed in variety of hematopoietic cell lines, resulting in the inhibition of iron induced apoptosis in these cells. Taken together, our data demonstrates for the first time that iron overload has direct suppressive effects on erythropoiesis. These effects are reversed by specific inhibitors of the TGFβ receptor I kinase and thus provide a preclinical rationale for these inhibitors in anemias associated with iron overload. Disclosures: No relevant conflicts of interest to declare.

A NUP98-HOXD13 Leukemic Fusion Gene Leads To Aberrant Actin Localization In Dysplastic Megakaryocytes

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2486-2486
Author(s):  
David L. Caudell ◽  
Benjamin Okyere ◽  
Jacob Cawley ◽  
Abdul Gafoor A. Puthiyaveetil ◽  
Bettina Heid
Keyword(s):  
Molecular Mechanisms ◽  
Conflicts Of Interest ◽  
Fusion Gene ◽  
Hematopoietic Malignancy ◽  
Glass Slides ◽  
Transmission Electron ◽  
Platelet Release ◽  
Actin Localization

Abstract Myelodysplastic syndrome (MDS) is a hematopoietic malignancy characterized by peripheral cytopenias due to bone marrow (BM) failure. Megakarypoiesis, megakaryocyte (MK) production, and platelet release are impaired in in some cases of MDS. Patients often have fewer, but larger circulating platelets, which have abnormal demarcation membrane systems (DMS); the DMS, which determines the number and size of platelets released, is dependent on actin formation. However, the precise role of actin during megakaryopoiesis is poorly understood. Transgenic mice that express the fusion gene NUP98-HOXD13 (NHD13) is a model for MDS and have dysplastic MKs in BM, and macro platelets in circulation. We hypothesized that expression of NHD13 disrupts actin localization during megakaryopoiesis resulting in reduced platelet release and macro platelet formation. To test the hypothesis, BM from wild type (WT) and NHD13 mice were flushed and cultured in media supplemented with Thrombopoietin for 5 days. Following in vitro propagation, MKs were harvested over a discontinuous gradient for downstream experiments. Sternums were also fixed in paraformaldehyde, stained with hematoxylin and eosin, and evaluated by light microscopy to analyze MK morphology in vivo. NHD13 BM contained many dysplastic MKs. Harvested MKs and BM cores from one femur were processed and analyzed by transmission electron microscopy (TEM) and the ultrastructural properties of the DMS detailed. TEM of MKs showed NHD13 leads to formation of an irregular DMS along with abnormal distribution of unusually large granules in MK cytoplasm. Cultured MKs were also cytospun onto glass slides, labeled with fluorescent-tagged F-actin and Myosin IIa and the cytoskeleton visualized by confocal microscopy. WT MKs in vitro had two phenotypes: (1) MKs with myosin and actin evenly dispersed in the cytoplasm and (2) MK with actin predominantly in the periphery of the cytoplasm. In contrast, transgenic MKs displayed only the former phenotype suggesting that actin localization is impaired in NHD13 MKs. Finally, MKs were stimulated with estrogen and adhered to fibrinogen matrices to determine their proplatelet formation functionality. Our results showed impaired proplatelets formation in NHD13 MKs. These data suggest that expression of NHD13 leads to aberrant actin localization leading to dysplastic MK differentiation and macro platelet release. Understanding molecular mechanisms of abnormal megakaryopoiesis in MDS is important as many MDS patients die of hemorrhagic complications. Further studies using this model system will provide a platform for translational research and should reveal potential therapeutic targets in MDS, leading to improved patient care/survival. Disclosures: No relevant conflicts of interest to declare.

A Whole Genome In Vivo Crispr Screen in Primary ALL Predicts Leukaemic Relapse

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2619-2619
Author(s):  
Katherine Dormon ◽  
Elda S Latif ◽  
Matthew Bashton ◽  
Deepali Pal ◽  
Matthew Selby ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Conflicts Of Interest ◽  
Gene Set Enrichment Analysis ◽  
Whole Genome ◽  
Competitive Situation ◽  
Nsg Mice ◽  
A Genome ◽  
Crispr Screen

Abstract Although paediatric acute lymphoblastic leukaemia (ALL) has a favourable prognosis, a number of cases will invariably relapse. One of the major problems associated with relapse is drug resistance, in particular to glucocorticoids, the mainstay of ALL treatment. Examining the underlying mechanisms is complicated by clonal heterogeneity within a patient and the potential impact of the leukaemic niche. To address mechanisms of drug resistance in a patient-relevant setting, we performed a genome-wide in vivo CRISPR screen in primary ALL material. To that end, we took advantage of primografted material from patient L707, who initially presented with a Dexamethasone (DEX) sensitive t(17;19) ALL, but relapsed 5 months after initial diagnosis. We transduced DEX sensitive presentation cells with the full genome GeCKOv2 CRISPR library, before transplantation into immunodeficient NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. Mice were subsequently treated with DEX by oral gavage (15mg/kg for 5 weeks, 10mg/kg thereafter). DNA from several engrafted sites in the mouse was extracted and PCR amplified before being sequenced on the Illumina HiSeq2500. Changes in pool complexity were analysed using MaGEcK software to determine which sgRNAs were significantly enriched or depleted. By far the most significantly enriched sgRNAs were those targeting NR3C1, the gene encoding the glucocorticoid receptor. In addition, two of the top five significantly depleted sgRNAs targeted the Plexins, PLXNA1 and PLXND1. Whilst PLXNA1 is expressed at low levels, PLXND1 is highly expressed and has been linked to dexamethasone resistance. Notably, the matched relapsed material from L707 was highly DEX resistant both in tissue culture and when transplanted into NSG mice. SNP 6.0 analysis revealed a 5q deletion in the relapse, spanning 5 genes including NR3C1. Whole genome sequencing showed this was comprised of 2 deletions both targeting NR3C1, with different breakpoints for each allele. The differential gene expression between the L707 presentation and relapse established that NR3C1 was the most significant of all the genes lost at relapse, based on gene set enrichment analysis (GSEA). This contrasts with many ALL cases, where one of the downstream effectors of apoptosis is lost as opposed to NR3C1. Growth of the relapse material in vivo and in vitro was slower than the presentation in a competitive situation, but with DEX treatment the relapse phenotype began to emerge with a small percentage of cells showing a heterozygous deletion of NR3C1. These combined data strongly suggest that the NR3C1 deletion is the main driver of DEX resistance in the L707 relapse. Moreover, it proves that our in vivo CRISPR screen predicted the leukaemic relapse. These results confirm NR3C1 deletion as a driver in glucocorticoid resistance and demonstrate the power of in vivo CRISPR screens to predict mechanisms of gain of drug resistance and subsequent relapse. The parallels that can be drawn between the relapse and the CRISPR screen are striking, giving the indication that the progression from presentation to relapse may follow the same path in a patient derived xenograft setting as it did in the patient. Disclosures No relevant conflicts of interest to declare.

Targeting apoptosis and autophagy by a novel bcl-2 inhibitor, GX15-070, in neuroblastoma.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. 10048-10048
Author(s):  
Herve Sartelet ◽  
Sonia Cournoyer ◽  
Anissa Addioui ◽  
Assila Belounis ◽  
Mona Beaunoyer ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Survival ◽  
Annexin V ◽  
High Sensitivity ◽  
Western Blots ◽  
Pediatric Tumor ◽  
Nsg Mice ◽  
Apoptotic Protein

10048 Background: Neuroblastoma (NB) is a frequent pediatric tumor with poor prognosis. The disregulation of the anti-apoptotic protein Bcl-2 is crucial for the tumoral development and chemoresistance. Autophagy is also implicated in tumor cell survival and chemoresistance. The aim of our study was to demonstrate the in vitro and in vivo therapeutic efficiency of GX 15-070, a Bcl-2 inhibitor, used alone and in combination with conventional drugs used in the treatment of NB and hydroxychloroquine (HCQ), a known autophagy inhibitor. Methods: Using 6 NB cell lines, cell viability (MTT) assays were done at progressively increased concentrations of GX 15-070 alone or in combination with cisplatin or with Z-VAD-FMK, a broad-spectrum caspase inhibitor. Apoptosis was tested by evaluating the cleavage of caspase 3 by western blots (WB) and the Annexin V/7-AAD staining studied by FACS. To assess if autophagy was modified by GX 15-070, the cleavage of LC3 protein was tested by WB and cell survival were tested with combination of GX 15-070 and HCQ. To verify the anti-tumor activity in vivo of GX 15-070, orthotopic injections were made on NSG mice treated with GX 15-070 alone and in combination with HCQ. Results: It was observed a high sensitivity of the NB cells to GX 15-070 with increase of cell death and a potential synergistic of this molecule when it’s combined with cisplatin or HCQ. This cell death was due to apoptosis and may also be inhibited by Z-VAD-FMK. GX 15-070 alone or associated to cisplatin increased the autophagy. The in vivo study showed that GX 15-070 treatment used alone or in combination with HCQ significantly decreased the size of the tumor. Conclusions: Our results support the interest of GX 15-070 in the treatment of NB alone or in combination with classical drugs. Our studies also support that activation of apoptosis associated with inhibition of autophagy have a synergistic potential against tumoral progression and must have to be considered in further mechanistic studies for the optimization of more efficient combined therapies in the treatment of NB.

Investigating Drug Resistance In Leukemia Initiating Cell Populations.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1570-1570
Author(s):  
Paraskevi Diamanti ◽  
Charlotte Victoria Cox ◽  
Allison Blair
Keyword(s):  
Oxidative Stress ◽  
Drug Resistance ◽  
Childhood Leukemia ◽  
Conflicts Of Interest ◽  
Annexin V ◽  
Cell Populations ◽  
Childhood All ◽  
Nsg Mice ◽  
Cd34 Cells

Abstract Abstract 1570 Outcomes for childhood leukemia have improved significantly in recent years with remission rates of over 98% and reported cure rates of 80% for standard risk cases. However, 20% of patients relapse due to failure to eradicate the disease. Further improvement in outcomes will require a better understanding of the biology of this malignancy and the mechanisms of drug resistance. Evidence that several leukemia subpopulations can initiate and maintain this disease in xenograft models and that some of these subpopulations are resistant to current therapeutic agents suggests that relapse may arise from these cells. Parthenolide (PTL), a sesquiterpene lactone compound, has been shown to cause apoptosis in malignant cells by inducing oxidative stress and by inhibiting NF-κB mediated cell survival. In this investigation we have assessed the effects of PTL on leukemia subpopulations in a cohort of childhood ALL cases from mixed prognostic subgroups. Cells from 15 B-ALL cases were stained with antibodies against CD34 and CD19, while CD34 and CD7 were used for 7 T-ALL cases. Cells were then sorted based on expression or lack of expression of the antibody combinations. Unsorted cells and the 4 sorted subpopulations from each type of leukemia were treated with 7.5 and 10 μM PTL for 18–24 hours. The effect of PTL on viability was studied by flow cytometry using Annexin V and Propidium iodide. In B-ALL cases, the CD34+/CD19- population was the least affected with 89.1±6.9% cells surviving PTL treatment. This was significantly higher than the unsorted cells and the other sorted populations (<53%; P<0.01). Most of the T-ALL cases (6/7) were classed as high risk by MRD analyses at day 28. Despite this, unsorted T-ALL cells were more responsive to PTL with only 29.7±12.8% surviving treatment. The CD34+/CD7- population was the least affected (59.9±13.3% viable cells). The functional capacity of the PTL treated unsorted cells and sorted populations was also assessed in vivo. NOD/SCID IL2Rγ null (NSG) mice were inoculated with untreated or PTL treated cells and the levels of engraftment after 10 weeks were compared. The results to date indicate that PTL treatment prevented engraftment of unsorted ALL cells. Mean engraftment levels of 65±20% CD45+ (range 29–99%) were observed using untreated cells while there was no detectable human cell engraftment with the PTL treated cells. This suggests PTL is more effective on unsorted ALL cells than the data from the short term apoptosis assays indicated. Engraftment was achieved using CD34- cells from 3 cases (73±29%, range 40–96%). However, no engraftment was observed when CD34- cells were treated with PTL. In contrast, the levels of engraftment observed with PTL treated CD34+/CD19- B-ALL cells were similar to or greater than those observed with the untreated counterparts (95±8% and 64±9% CD45+ respectively, P≤0.07). The levels of engraftment observed with CD34+/CD7- T-ALL cells were reduced with PTL treatment from 67±21% to 12±9% CD45+ (P≤0.03) but not eliminated. Subsequently, we investigated the mechanisms for this apparent resistance to PTL in the primitive cell populations. PTL has been associated with induction of oxidative stress, activation of p53 and inhibition of NF-κB in AML and CLL. We used confocal microscopy to investigate whether NF-κB is constitutively expressed in ALL cases and to evaluate the effect of PTL on the phosphorylation of NF-κB. Three B-ALL and 3 T-ALL cases, where the unsorted populations had been affected by PTL while the respective CD34+/CD19- and CD34+/CD7- populations were more resistant, were investigated. Cells were stained with anti-phospho-p65 polyclonal antibody and Alexa fluor 488. NF-κB was constitutively activated in all cases. There was evidence of decreased phosphorylation in unsorted PTL treated cells indicating inhibition of NF-κB. However, in the phenotypically primitive cells there was no difference in the phosphorylation levels compared to untreated cells or phosphorylation was increased. This suggests NF-κB was not inhibited, which could explain the observed resistance of these leukemia populations to PTL. These data demonstrate that some leukemia initiating cell populations in childhood B-ALL and T-ALL are resistant to PTL. A more thorough understanding of these leukemia initiating cell populations and their mechanisms of resistance will be required for the development of more effective therapies. Disclosures: No relevant conflicts of interest to declare.

Combined Targeting of BCR-ABL and JAK2 with ABL and JAK2 Inhibitors Is Effective Against CML Patients' Leukemic Stem/Progenitor Cells.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3404-3404
Author(s):  
Donna DeGeer ◽  
Paolo Gallipoli ◽  
Min Chen ◽  
Ivan Sloma ◽  
Heather Jorgensen ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Kinase Inhibitor ◽  
Conflicts Of Interest ◽  
Integration Site ◽  
Single Agent ◽  
K562 Cells ◽  
High Rate ◽  
Jak2 Inhibitors

Abstract Abstract 3404 Imatinib mesylate (IM) is a tyrosine kinase inhibitor (TKI) that induces clinical responses in most chronic myeloid leukemia (CML) patients. Nevertheless, early relapses and later emergence of IM-resistant disease pose serious concerns for many. The inadequacies of IM therapy are due, at least in part, to the unique properties of CML stem/progenitor cells that make them generally less responsive to IM and, indeed, other TKIs, and also confer on them a genetic instability that leads to a high rate of formation of BCR-ABL mutants. Improved treatment approaches to prevent the development of resistant subclones by targeting other key molecular elements active in CML stem/progenitor cells are thus clearly needed. One candidate is a complex that forms in CML stem/progenitor cells between the oncoproteins encoded by AHI-1 (Abelson helper integration site 1), BCR-ABL and the JAK2 kinase. This complex contributes to the transforming activity of BCR-ABL both in vitro and in vivo and also plays a role in the IM response/resistance of primary CML stem/progenitor cells. We now describe the results of experiments designed to test the ability of ABL and JAK2 inhibitors to block the activity of this protein complex in CML cells. K562 cells engineered to stably overexpress AHI-1 showed a significantly reduced sensitivity to both IM (at 1 and 5 μM) and TG101209, a JAK2 inhibitor, (at 0.5 and 1 μM), as determined by assays for cell viability, apopotosis, and colony-forming activity. K562 cells engineered to suppression AHI-1 showed an opposite effect, with a heightened sensitivity to IM at concentrations as low as 1 μM. In addition, IM together with TG101209 was more effective at killing AHI-1-overexpressing K562 cells, IM-resistant K562 cells and IM-resistant T315I-mutant cells than either treatment alone. Western blot and co-IP experiments demonstrated a significant reduction of p-BCR-ABL, p-JAK2 and p-STAT5 in cells treated with IM plus TG101209 compared to cells treated with IM or TG101209 alone. Importantly, treatment with 5 μM IM, 150 nM dasatinib (DA) or 5 μM nilotinib (NL) in combination with 100 nM TG101209 caused a significantly greater reduction in the viability of primary CD34+CD38− and CD34+CD38+ CML cells when these responses were compared to any of the TKIs or TG101209 alone (~2-4 fold, n=3). Apoptotic cells at 72 hours were also significantly increased for all drug combinations compared to single agent treatments (40%-52% for the combinations vs 15%-18% for the single agents). CFSE tracking analysis of cell division in these cells further demonstrated additive anti-proliferative activity from the TKI plus TG101219 combinations, although some rare undivided cells were not eliminated. Nevertheless, exposure of CD34+ CML cells from IM-nonresponders (n=4) to TG101209 plus IM or DA did cause a greater inhibition (81% and 85%) of patients' colony-forming cells as compared to the same cells treated with the combination of IM plus DA only, or IM or DA only (60%, 41% and 50% inhibition, p<0.05). Long-term culture-initiating cell assays were undertaken to compare the effect of these combination treatments versus the effects of TKIs or TG101209 alone on very primitive CML cells. The results again showed a more significant reduction of these cells treated with the combination (n=3). Intracellular staining revealed a greater reduction in the levels of p-CrKL and p-STAT5 in CD34+ CML cells treated for 24 hours with the combination of TKIs plus TG101219 as compared to single TKI-treated cells (~44% vs 65% for p-CrKL and 36% vs 57% for p-STAT5, n=3). Strikingly, the combination treatment produced an even greater inhibition of both p-CrKL and p-STAT5 after 72 hours while p-CrKL was almost fully reactivated with TKIs alone (~29% vs 89% for p-CrKL and 23% vs 50% for p-STAT5). These results point to the possibility of achieving improved therapeutic outcomes in CML patients by simultaneously targeting both BCR-ABL and JAK2 activities in the critical TKI-insensitive CML stem/progenitor reservoir. Disclosures: No relevant conflicts of interest to declare.

Lycorine Modulates the Expression of p21 Via a p53-Independent Pathway in HL-60 Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4297-4297
Author(s):  
Jing Liu ◽  
Shu-Ling Wang ◽  
Lin Fang ◽  
Mao Ye ◽  
Zhi-Wei Sun ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Kinase Inhibitor ◽  
Conflicts Of Interest ◽  
Fold Increase ◽  
Alternative Pathways ◽  
Cyclin Dependent Kinase Inhibitor ◽  
M Phase ◽  
Induced Apoptosis

Abstract Abstract 4297 Leukemia is one of the most life-threatening cancers today, and acute promyelogenous leukemia is a common type of leukemia. We have previously shown that lycorine, a natural alkaloid extract from Amaryllidaceae, exhibited anti-leukemia effects in vitro and in vivo. Lycorine treatment of HL-60 cell arrested cell cycle at G2/M phase and induced apoptosis. In the present study, we sought to explore the molecular mechanisms for the anti-leukemia action of lycorine. Gene chip analysis revealed that lycorine treatment of HL-60 cells induced more than 9 fold increase of p21, a cyclin-dependent kinase inhibitor, whose expression is mainly regulated by p53. Since HL-60 cells are p53 null, the above findings suggest that lycorine activates p21 expression through p53-independent pathway. To further explore the alternative pathways for the activation of p21 induced by lycorine, we examined the effect of lycorine on the expression of Rb, pRb, E2F, c-Myc and HDACs which have shown to regulate p21 expression. We show that expression of pRb (ser780) and c-Myc was down-regulated, Rb and E2F were up-regulated, while the expression of HDAC1 and HDAC3 was not changed. Together these findings suggest that lycorine exerts its anti-leukemia effect by activating p21 expression via pRb/E2F and c-Myc pathways. Disclosures: No relevant conflicts of interest to declare.

Assessing CD97 and CD99 As Markers of Leukaemia Initiating Cells in Paediatric ALL

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1882-1882 ◽  
Author(s):  
Charlotte Victoria Cox ◽  
Paraskevi Diamanti ◽  
Allison Blair
Keyword(s):  
Functional Capacity ◽  
Conflicts Of Interest ◽  
Residual Disease ◽  
Survival Rates ◽  
In Vivo Studies ◽  
Nsg Mice ◽  
Early Treatment Response ◽  
Current Therapies

Abstract Abstract 1882 Overall survival rates in paediatric acute lymphoblastic leukaemia (ALL) have dramatically improved but around 20% do not respond to current therapies and subsequently relapse. Leukaemia initiating cells (LIC) are the topic of much investigation, as these cells can self-renew and may have the potential to cause relapse. It has been shown that multiple subpopulations of ALL cells have the ability to initiate the disease in immune deficient mouse models. Therefore, treatment should be targeted at all cells with this capacity, if the disease is to be eradicated. Minimal residual disease (MRD) detection is an invaluable tracking tool to assess early treatment response and recent studies have highlighted potential markers that may improve the sensitivity of MRD detection by flow cytometry. CD97 and CD99 are two markers which were over expressed in paediatric ALL. Incorporating these markers into investigations of LIC may allow discrimination of leukaemia cells from normal haemopoietic stem cells (HSC). In this study we evaluated the expression of CD34 in combination with CD97 in B cell precursor (BCP) ALL cases and CD99 in T-ALL cases and subsequently assessed the functional capacity of the sorted subpopulations in vitro and in vivo. Ten ALL samples (6 B-ALL & 4 T-ALL) with a median age 7 years (range 2–15 years) were studied. One B-ALL case and 3 T-ALL cases were considered high risk by molecular assessment of MRD at day 28 of treatment. Flow cytometric analyses of the ALL samples and 8 normal haemopoietic cell samples demonstrated that both CD97 and CD99 were over expressed in ALL patients (78.9±14.8% & 76.4±32.8%, respectively) when compared to normal haemopoietic cells (14.1±25.4%; p=0.001, 47.1±10%; p=0.03, respectively). Cells were sorted for expression/lack of expression of these markers and proliferation of the sorted cells was assessed in suspension culture over a 6 week period. In the B-ALL patients the CD34+/CD97+ subpopulation represented the bulk of leukaemia cells (65.2±32.1%), the CD34−/CD97+ the smallest fraction (3.3±2.4%) with the CD34+/CD97− and CD34−/CD97− subpopulations representing 21.1±31.5% and 10.5±5.8% of cells, respectively. When the functional capacity of these subpopulations was assessed in vitro greatest expansion was observed in cells derived from CD34+/CD97− subpopulation (2–173 fold) from 9.4×103 at initiation up to 1.5×106 cells at week 6. Expansion was also observed, to a lesser extent in the CD34−/CD97− subpopulation (3.4–28 fold) from 8×103 up to 1.4×106 cells. No expansion was observed in cultures of CD34+/CD97+ and CD34−/CD97− subpopulations but cells were maintained throughout the culture period. These sorted subpopulations were also inoculated into NOD/LtSz-SCID IL-2Rγc null (NSG) mice to evaluate repopulating capacity. To date, engraftment has been achieved with 3 subpopulations; CD34+/CD97+ (3–28.8% CD45+), CD34+/CD97− (0.5–25.5% CD45+) and CD34−/CD97+ (23.8% CD45+) cells. When the functional capacity of T-ALL cases was assessed the CD34+/CD99+ subpopulation represented the bulk of cells at sorting (51.87±47.2%), the CD34+/CD99- subpopulation was the smallest (0.9±0.8%) and the CD34−/CD99+ and CD34−/CD99− subpopulations represented 32.1±38.9% and 27.2±33.4% of cells, respectively. Greatest expansion was observed in cultures of CD34+/CD99- cells (4.6–1798 fold) from 7.5×103 up to 2.6×106 cells at week 6. The other 3 subpopulations expanded to a lesser extent (1.3–216 fold) from 5×103 up to 1.8×106 cells. When the functional capacity of these cells was assessed in NSG mice, engraftment was achieved in all subpopulations; CD34+/CD99+ (87–90.5% CD45+), CD34+/CD99− (1.5–84.9% CD45+), CD34−/CD99+ (31.3–98.6% CD45+) and CD34−/CD99− (3–92.9% CD45+). In some cases, cells recovered from BM of NSG inoculated with CD99− cells had high expression of CD99, typical of the patient samples at diagnosis, indicating that the inoculated CD99− cells had differentiated in vivo. Studies are ongoing to assess the self-renewal capacity of these subpopulations by serial transplantation. The findings to date indicate that targeting CD97 and CD99, either alone or in combination with CD34 would not eliminate all cells with the capacity to initiate and maintain B-ALL and T-ALL, respectively. Further developments in therapy may require targeting leukaemogenic pathways, rather than only cell surface markers to improve survival outcome in paediatric ALL. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Modulated Electro-Hyperthermia Resolves Radioresistance of Panc1 Pancreas Adenocarcinoma and Promotes DNA Damage and Apoptosis In Vitro

2020 ◽  
Vol 21 (14) ◽  
pp. 5100 ◽  
Author(s):  
Gertrud Forika ◽  
Andrea Balogh ◽  
Tamas Vancsik ◽  
Attila Zalatnai ◽  
Gabor Petovari ◽  
...  
Keyword(s):  
Kinase Inhibitor ◽  
Chromatin Condensation ◽  
Annexin V ◽  
Therapy Resistance ◽  
In Vivo Model ◽  
Tumor Growth Inhibition ◽  
Dna Double Strand Breaks ◽  
Cell Fractions

The poor outcome of pancreas ductal adenocarcinomas (PDAC) is frequently linked to therapy resistance. Modulated electro-hyperthermia (mEHT) generated by 13.56 MHz capacitive radiofrequency can induce direct tumor damage and promote chemo- and radiotherapy. Here, we tested the effect of mEHT either alone or in combination with radiotherapy using an in vivo model of Panc1, a KRAS and TP53 mutant, radioresistant PDAC cell line. A single mEHT shot of 60 min induced ~50% loss of viable cells and morphological signs of apoptosis including chromatin condensation, nuclear shrinkage and apoptotic bodies. Most mEHT treatment related effects exceeded those of radiotherapy, and these were further amplified after combining the two modalities. Treatment related apoptosis was confirmed by a significantly elevated number of annexin V single-positive and cleaved/activated caspase-3 positive tumor cells, as well as sub-G1-phase tumor cell fractions. mEHT and mEHT+radioterapy caused the moderate accumulation of γH2AX positive nuclear foci, indicating DNA double-strand breaks and upregulation of the cyclin dependent kinase inhibitor p21waf1 besides the downregulation of Akt signaling. A clonogenic assay revealed that both mono- and combined treatments affected the tumor progenitor/stem cell populations too. In conclusion, mEHT treatment can contribute to tumor growth inhibition and apoptosis induction and resolve radioresistance of Panc1 PDAC cells.

scholarly journals Dasatinib Is Effective in the Treatment of Mice Models with Immune Thrombocytopenia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1456-1456
Author(s):  
Tadashi Shimoyama ◽  
Yoshiaki Okano ◽  
Yukiteru Fujishima ◽  
Tatsuo Oyake ◽  
Shugo Kowata ◽  
...  
Keyword(s):  
Immune Thrombocytopenia ◽  
Kinase Activity ◽  
Kinase Inhibitor ◽  
Conflicts Of Interest ◽  
Fc Receptor ◽  
Src Family Kinase ◽  
Igg Antibody ◽  
Platelet Counts

Abstract Background: Immune thrombocytopenia (ITP) is an autoimmune disease in which anti-platelet antibody (APA) is produced. APA-coated platelets are captured and phagocytized by macrophages in the spleen. Recent study revealed that spleen tyrosine kinase (Syk) inhibitor is effective in the treatment of ITP, because Syk phosphorylation is the key step of the phagocytosis by macrophages. Activated Fc receptor signal transduction is initiated by phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) tyrosine residues by SRC family kinases. Recruitment of Syk to dually phosphorylated ITAMs triggers the activation of Syk. To prove the hypothesis that the inhibition of SRC family kinase induces the decreased phosphorylation of Syk, resulting in decreased phagocytosis by macrophages, a SRC family kinase inhibitor, dasatinib, was used in the experiments. Methods, Results and Discussion: In vitro study; Murine macrophage cell line, RAW, was incubated with APA coated murine platelets for 30 minutes. Phagocytosis by RAW was significantly decreased with dasatinib (100nM, p<0.01), indicating SRC family kinase activity is required for efficient phagocytosis. Phosphorylated Syk was decreased in RAW, incubated with anti-Fc receptor antibody (rat IgG) and anti-rat IgG antibody with dasatinib (100nM), shown in the Western blot analysis (Figure 1). These results suggest that Syk phosphorylation is the key step in phagocytosis. In vivo study; (1) Three hours before APA intra-peritoneal injection, dasatinib (2.5mg/kg) was oral-administrated. Six hours after APA injection, platelet counts were measured. The platelet counts were 366 ± 164 x109/L with dasatinib (n=4, mean ± SD) and 114 ± 51 x109/L without dasatinib (n=4)(P=0.026)(Figure 2). (2) Osmotic pump, filled with APA, were inserted in murine intra-peritoneal cavity and dasatinib (2.5mg/kg) was oral-administered once daily for 7 days. The platelet counts were 499 ± 98 x109/L with dasatinib (n=4, mean ± SD) and 82 ± 131 x109/L without dasatinib (n=4) at day 7 (p<0.0022) (Figure 3). These results strongly suggest that dasatinib inhibit the phagocytosis in vivo. Conclusion: Dasatinib inhibits phosphorylation of Syk, inducing decreased phagocytosis of APA-coated platelets via decreased SRC family kinase activity. These findings reveal that SRC family kinase controls the efficiency of phagocytosis in part through the regulation of Syk function. Dasatinib might be effective in the treatment of ITP. Disclosures No relevant conflicts of interest to declare.

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