scholarly journals Role of kif2c, A Gene Related to ALL Relapse, in Embryonic Hematopoiesis in Zebrafish

2020 ◽  
Vol 21 (9) ◽  
pp. 3127 ◽  
Author(s):  
Chang-Kyu Oh ◽  
Ji Wan Kang ◽  
Yoonsung Lee ◽  
Kyungjae Myung ◽  
Mihyang Ha ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Prognostic Markers ◽  
Lymphoblastic Leukemia ◽  
Zebrafish Embryos ◽  
Related Gene ◽  
Binding Partner ◽  
First Time ◽  
Novel Therapeutic

Relapse of acute lymphoblastic leukemia (ALL) is dangerous and it worsens the prognosis of patients; however, prognostic markers or therapeutic targets for ALL remain unknown. In the present study, using databases such as TARGET, GSE60926 and GSE28460, we determined that KIF2C and its binding partner, KIF18B are overexpressed in patients with relapsed ALL compared to that in patients diagnosed with ALL for the first time. As 50% of the residues are exactly the same and the signature domain of KIF2C is highly conserved between human and zebrafish, we used zebrafish embryos as a model to investigate the function of kif2c in vivo. We determined that kif2c is necessary for lymphopoiesis in zebrafish embryos. Additionally, we observed that kif2c is not related to differentiation of HSCs; however, it is important for the maintenance of HSCs as it provides survival signals to HSCs. These results imply that the ALL relapse-related gene KIF2C is linked to the survival of HSCs. In conclusion, we suggest that KIF2C can serve as a novel therapeutic target for relapsed ALL.

Effective Induction of Apoptosis by Mistletoe Plant Extracts in an Acute Lymphoblastic Leukemia Model.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1880-1880
Author(s):  
Georg Seifert ◽  
Patrick Jesse ◽  
Aram Prokop ◽  
Tobias Reindl ◽  
Stephan Lobitz ◽  
...  
Keyword(s):  
Cell Death ◽  
Body Weight ◽  
Acute Lymphoblastic Leukemia ◽  
Lymphoblastic Leukemia ◽  
The Body ◽  
Induction Of Apoptosis ◽  
First Time ◽  
Over Time

Abstract Mistletoe (Viscum album) is one of the most used alternative cancer therapies applied as monotherapy or in combination with conventional therapies. Anti-tumor effects of mistletoe (MT) extracts were related to cytostatic and immunomodulatory effects observed in vitro. Aqueous MT extracts contain the three mistletoe lectins I, II and III as one predominant group of biologically active agents. The MT lectins inhibit protein biosynthesis by inactivating the 60S ribosomal subunit. Mistletoe lectin-I (ML-I) is one important apoptosis inducing compound. It is a heterodimer that consists of a cytotoxic A-chain (ribosome inactivating protein, RIP type 1) linked by a carbohydrate binding B-chain for cellular lectin uptake. However, although MT is widely used, there is a lack of scientific preclinical and clinical data. Here, we describe for the first time efficacy and mechanism of MT extracts against lymphoblastic leukemia in vitro and in vivo. For this purpose, we first investigated both the cytotoxic effect and mechanism of action of two standardized aqueous MT extracts (MT obtained from fir trees (MT-A); MT obtained from pine trees (MT-P)) and isolated ML-I, in three human acute lymphoblastic leukemia (ALL) cell lines (NALM-6, sup-B-15 and REH). MT-A, MT-P and ML-I clearly inhibited cell proliferation as determined by LDH reslease assays at very low concentrations (ML-I LD50 from 0,05 ng/ml to 10 ng/ml depending on the host tree) with MT-P being the most cytotoxic extract. The mechanism of cell death was determined by DNA-fragmentation assays. These indicated dose dependent induction of apoptosis as the main mechanism of cell death. Finally, we evaluated the efficacy of MT-A and MT-P in an in vivo SCID-model of pre-B ALL (NALM-6). For this purpose, mice (n=8/group) were injected i.v. with 1 × 106NALM6 cells and treated by intraperitoneal injections four times per week for 3 weeks (day 1–4; 7–11; 14–18) at varying doses (1, 5 and 50 mg/Kg (plant weight/body weight)). Mice (n=8) treated with PBS and cyclophosphamide (100 mg/kg, once on day 1) were used as negative and positive controls, respectively. Toxicity, peripheral blood counts, bodyweight and survival was determined over time. Interestingly, both MT extracts in all tested concentrations significantly improved survival (up to 55,4 days) in contrast to controls (34,6 days). Furthermore, no hematologic side effects were observed from this treatment as indicated by completely stable blood counts. Also the body weight of treated animals remained stable over time indicating a complete absence of systemic toxicity in the selected dose range. In summary, we demonstrate for the first time efficacy and mechanism of MT extracts against ALL in vitro and in vivo and hereby provide an important base line for the design of clinical trials with these compounds.

Therapeutic Efficacy of Natural Compounds From Viscum Album l. In Acute Lymphoblastic Leukemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3261-3261
Author(s):  
Catharina I. Delebinski ◽  
Sebastian Jaeger ◽  
Kristin Kemnitz-Hassanin ◽  
Arend von Stackelberg ◽  
Günter Henze ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Scid Mouse ◽  
Lymphoblastic Leukemia ◽  
Viscum Album ◽  
Mitochondrial Potential ◽  
Scid Mouse Model ◽  
Mistletoe Extracts ◽  
First Time

Abstract Abstract 3261 Viscum album L. (mistletoe) is one of the most widely used complementary cancer therapies. Due to their low solubility, aqueous extracts contain hardly any triterpenes which are known to possess anti-tumoral properties. Using cyclodextrins it was possible to solubilisate mistletoe triterpenes (mainly oleanolic acid (OA)) and achieve a plant extract with high levels of OA and mistletoe lectins (ML). In the present study, we determined for the first time the effect of clearly defined mistletoe extracts against human acute lymphoblastic leukemia (ALL) in vitro and in vivo. These mistletoe extracts contain either lectins (aqueous extract, viscum) or cyclodextrin solubilised triterpenes (STE) such as oleanolic - and betulinic acid and combinations thereof (viscumTT). We used the C.B-17/SCID mouse model and tested efficacy and mechanisms of the treatment with these preparations in vitro and in vivo. The human leukemia cell line NALM-6 was incubated with increasing concentrations of mistletoe preparations (10-60 μg/ml OA; 0.8–8 ng/ml ML) and tested for their cytotoxicity in vitro. Apoptosis was determined using mitochondrial potential, DNA fragmentation and Annexin/PI assays. In vivo efficacy was determined in the C.B-17/SCID mouse model. For this purpose, 1×106 NALM-6 cells were injected IV into groups of C.B-17/SCID mice (n=8) and STE extracts were administered three times per week for 14 days by intraperitoneal (IP) injection. Viscum album L. extracts inhibited cell proliferation and show cytotoxic properties in vitro. The highest level of apoptosis with a decrease of the mitochondrial potential was observed with STE preparation at a concentration of 50 μg/ml OA and for lectin-treated cells for 4.7 ng/ml (IC50). To exclude an unwanted cell death via necrosis, LDH release was measured after 4h of incubation with different doses and extracts of Viscum album L without significant LDH release. Based on these data, we investigated the effect of Viscum album L. extracts in vivo. For this purpose 40 mg/kg/day oleanolic acid (STE), 3 μg/kg/day lectin (viscum) or a combination thereof (viscumTT) were administered IP. In line with the in vitro results, mice treated with viscumTT showed a significant longer survival. Mice receiving PBS had a mean survival time of 38 days whereas mice treated with viscumTT had a mean survival of 50,5 days (p=0,005). In summary, we demonstrate for the first time that either solubilised triterpenes or lectins and combinations thereof induce dose- and time-dependent apoptosis in the ALL cell line NALM-6. Based on the in vivo data we believe that triterpene containing Viscum album L. extracts may possess an impressive therapeutic potential. Thus, our investigations provide an important base line for the design of further experimental studies and clinical trials to investigate the effects of individual components and potential synergisms in ALL. Disclosures: No relevant conflicts of interest to declare.

Role of SLC12A10.2, a Na-Cl cotransporter-like protein, in a Cl uptake mechanism in zebrafish (Danio rerio)

2009 ◽  
Vol 296 (5) ◽  
pp. R1650-R1660 ◽  
Author(s):  
Yi-Fang Wang ◽  
Yung-Che Tseng ◽  
Jia-Jiun Yan ◽  
Junya Hiroi ◽  
Pung-Pung Hwang
Keyword(s):  
Specific Inhibitor ◽  
In Vivo Model ◽  
Zebrafish Embryos ◽  
Uptake Mechanism ◽  
Ion Regulation ◽  
Specific Expression ◽  
Net Uptake ◽  
First Time

The thiazide-sensitive Na+-Cl− cotransporter (NCC), a member of the SLC12 family, is mainly expressed in the apical membrane of the mammalian distal convoluted tubule (DCT) cells, is responsible for cotransporting Na+ and Cl− from the lumen into DCT cells and plays a major role in the mammalian renal NaCl reabsorption. The NCC has also been reported in fish, but the functional role in fish ion regulation is yet unclear. The present study used zebrafish as an in vivo model to test the hypothesis of whether the NCC plays a role in Na+ and/or Cl− uptake mechanisms. Four NCCs were cloned, and only one of them, zebrafish (z) slc12a10.2 was found to predominately and specifically be expressed in gills. Double in situ hybridization/immunocytochemistry in zebrafish skin/gills demonstrated that the specific expression of zslc12a10.2 mRNA in a novel group of ionocytes differed from those of the previously-reported H+-ATPase-rich (HR) cells and Na+-K+-ATPase-rich (NaR) cells. Gill mRNA expression of zslc12a10.2 was induced by a low-Cl environment that stimulated fish Cl− influx, while a low-Na environment suppressed this expression. Incubation with metolazone, a specific inhibitor of the NCC, impaired both Na+ and Cl− influx in 5-day postfertilization (dpf) zebrafish embryos. Translational knockdown of zslc12a10.2 with a specific morpholino caused significant decreases in both Cl− influx and Cl− content of 5-dpf zebrafish embryos, suggesting that the operation of zNCC-like 2 results in a net uptake of Cl− in zebrafish. On the contrary, zslc12a10.2 morphants showed increased Na+ influx and content that resulted from upregulation of mRNA expressions of Na+-H+ exchanger 3b and carbonic anhydrase 15a in HR cells. These results for the first time provide in vivo molecular physiological evidence for the possible role of the NCC in the Cl− uptake mechanism in zebrafish skin/gills.

scholarly journals Targeting Nucleolin for Reversal of Chemotherapy Resistance in Acute Lymphoblastic Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5058-5058
Author(s):  
Jianda Hu ◽  
Yanxin Chen ◽  
Zhengjun Wu ◽  
Lingyan Wang ◽  
Jingjing Wen ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Acute Lymphoblastic Leukemia ◽  
Drug Sensitivity ◽  
De Novo ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Mrna Level ◽  
Chemotherapy Resistance

Chemotherapy resistance is considered to be the principal cause of ineffective treatment in acute lymphoblastic leukemia (ALL). Nucleolin (NCL) is high expression andplays oncogenic roles in most cancers. However, less research on the role of NCL in hematologic malignancies was noted. Our previous studies have showed that overexpression of NCL was associated with worse prognosis in the patients with acute leukemia and NCL expressionwashigher in resistant HL-60/ADR than in sensitive HL-60 cells. The potential mechanisms of NCL in chemotherapy resistance have yet to be revealed. Here we presented that expression of NCL was associated positively with chemotherapy resistance and poor prognosis in ALL. Overexpressed NCL at both mRNA and protein level was relevant to a poorer overall survival (OS) and relapse free survival (RFS), indicating NCL as an independent prognostic marker in ALL. mRNA level of NCL in de novo ALL was quantitatively higher than in complete remission(CR) status, and refractory/relapse ALL had the highest level. Upon above clinical data, we further investigated the mechanism(s) by which NCL regulated drug resistance in ALL cells. Remarkably, NCL expression was higher in resistant ALL cells relative to sensitive parental cells. When treated with ADM, NCL level was decreased in sensitive parental cells while unchanged in resistant cells. Overexpressing NCL suppressed drug sensitivity, altered drug effluxion and decreased intracellular drug accumulation, while inhibition of NCL led to a completely reversed appearance, more intracellular Adriamycin(ADM) mean fluorescence intensity (MFI) and percentage of ADM accumulated cells population. Overexpression of NCL increased significantly the IC50 of ADM. The IC50 of ADM on Jurkat-NCL-overexpression(OE), Jurkat-NCL-knockdown(KD), Molt-4-NCL-OE, Molt-4-NCL-KD, Nalm-6-NCL-OE, Nalm-6-NCL-KD were 1.362±0.271μg/ml, 0.077±0.010μg/ml, 4.863±0.733μg/ml, 0.081±0.018μg/ml, 0.237±0.042μg/ml and 0.046±0.002μg/ml, respectively (P <0.05). Involvement of ATP-binding cassette (ABC) transporters was proved in NCL mediated drug resistance. Silencing NCL resulted in a decrease of P-gp, MRP1, LRP and BCRP in ALL cells, and NCL overexpression increased the MRP1, LRP and BCRP. The Akt/mTOR and ERK signaling pathways were involved in this procedure. Notably, co-IP assays confirmed the NCL-Ras, NCL-ERK and NCL-BCRP interaction. For intervention study, aptamer AS1411, a NCL inhibitor, could reduce drug resistance in ALL cell lines and primary ALL cells.Moreover, AS1411 treatment decreased BCRP protein expression. Furthermore, the ALL leukemia models that nude mice engrafted with Nalm-6 cells and NCG mice engrafted with Luc+ Nalm-6 cells were established, then treated with ADM plus AS1411 or control CRO for comparison drug sensitivity and survival. Growth of subcutaneous xenograft tumors was inhibited in those treated with AS1411 or ADM, compared to their respective controls treated with CRO or PBS. The stronger inhibition effect was observed in those treated with AS1411 combined with ADM. For Luc+Nalm-6 derived ALL model, leukemia progression was suppressed in mice treated with AS1411 and AS1411 combined with ADM. AS1411and ADM, especially combination of AS1411 and ADM, could improve survival of the leukemic mice compared to those treated with PBS. The results showed that NCL targeted by AS1411 sensitized ADM treatment and prolonged survival in vivo. In summary, our findings revealed NCL as a survival predictor and the novel role of NCL in ALL chemo-resistance. NCL may be a potential target for improving outcome in ALL. Disclosures No relevant conflicts of interest to declare.

L-arginine depletion by PEG-arginase I, a new potential therapy for acute lymphoblastic leukemia.

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. 6580-6580
Author(s):  
Ofelia Crombet Ramos ◽  
Claudia Hernandez ◽  
Kevin Morrow ◽  
John T. Cole ◽  
Paulo Rodriguez
Keyword(s):  
Cell Proliferation ◽  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
De Novo ◽  
Remission Rate ◽  
Lymphoblastic Leukemia ◽  
Arginase I

6580 Background: Advances in therapies have resulted in an overall complete remission rate of approximately 85% for childhood acute lymphoblastic leukemia (ALL). In contrast, the overall remission rate of adults with leukemia continues to be poor, only about 40% in cases of T cell-ALL (T-ALL). Therefore, it is imperative to generate new therapies that alone or in combination with other treatments could potentially increase the percentages of complete responders or be used to treat the refractory ALL population. Our published results show that a pegylated form of human arginase I (peg-Arg I) prevented T-ALL cell proliferation in vitro and in vivo through the induction of tumor cell apoptosis. Interestingly, the anti-leukemic effects induced by peg-Arg I did not affect the anti-tumor activity of normal T cells, suggesting an anti-tumor specific effect. Our hypothesis states that peg-Arg I has an anti-tumoral effect on B-ALL and T-ALL cells in vitro and that the sensitivity of ALL cells to peg-Arg I depends on their expression of argininosuccinate synthase (ASS) and their ability to produce L-arginine de novo from citrulline. Methods: Malignant T cell proliferation was tested using nonradioactive cell proliferation yellow tretrazolium salt kit. Apoptosis studies were based on the expression of annexin V. Western blot assays were conducted to determine enzymatic expression in different cell lines. Results: The results of our in vitro experiments showed that peg-Arg I had a pro-apoptotic and anti-proliferattive effect on B-ALL cells similar to the one previously seen on T-ALL cells. These effects can be overcome in cell lines able that express ASS and therefore to produce L-arginine de novo. Conclusions: Our results suggest the role of ASS in the ALL-apoptosis induced by peg-Arg-I. Our next steps include: _Understand why ASS-expressing ALL cells do not undergo apoptosis when cultured with peg-Arg-I_Determine the role of ASS in the anti-leukemic effect induced by peg-Arg-I in vivo. Completion of this research is expected to lead to a better understanding of how peg-Arg-I kills ALL cells and could provide the foundation for a novel therapy for ALL patients.

scholarly journals Identification of the Energy Stress Sensor AMPK As Therapeutic Target in Acute Lymphoblastic Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2771-2771
Author(s):  
Lai N Chan ◽  
Jaewoong Lee ◽  
Kadriye Nehir Cosgun ◽  
Huimin Geng ◽  
Gang Xiao ◽  
...  
Keyword(s):  
Cell Death ◽  
Acute Lymphoblastic Leukemia ◽  
B Cell ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Lymphoblastic Leukemia ◽  
Cell Lineage ◽  
Glycolytic Activity ◽  
Novel Therapeutic

Abstract Background and Hypothesis: While often transformed by the same oncogenes, biological and clinical characteristics of B-cell lineage and myeloid leukemias markedly differ. For instance, BCR-ABL1 tyrosine kinase drives both chronic myeloid leukemia (CML) and B cell lineage Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL). While the majority of CML patients achieve long-term disease-free survival under treatment, Ph+ ALL patients invariably relapse within months after initial remission. Here, we investigated whether the distinct characteristics of Ph+ ALL and CML have a metabolic basis and identified the metabolic sensor LKB1 and its substrate AMPK as novel therapeutic targets in pre-B ALL. Results: Metabolic measurements revealed strikingly higher AMP:ATP ratios with concomitant decreases in ATP production in patient-derived Ph+ ALL cells when compared to CML cells. These findings indicate a state of chronic energy depletion in pre-B ALL cells. Energy deficit activates the LKB1-AMPK energy sensor pathway to stimulate glucose uptake to restore ATP levels. Notably, LKB1 levels and activity of its substrate AMPKα were higher in patient-derived Ph+ ALL cells compared to CML cells. To study the consequences of inducible deletion of Lkb1, murine BCR-ABL1-driven myeloid lineage (CML) and B cell precursor (Ph+ ALL) Lkb1fl/fl leukemia cells were generated. We found that Lkb1-deletion increased glycolysis, ATP levels and proliferation in myeloid leukemia, consistent with the common notion that LKB1 is an established tumor suppressor. On the contrary, loss of Lkb1 function resulted in diminished glycolytic activity, impaired mitochondrial functions and cell death in pre-B ALL cells. C/EBPa-mediated reprogramming of B-cell into myeloid identity reversed the detrimental effects of Lkb1-deletion, restoring glycolysis, energy levels and survival of B→ myeloid reprogrammed cells. To study Lkb1 early in B cell lineage in vivo, Lkb1fl/fl mice were crossed with Mb1-Cretg/+ mice. Loss of Lkb1 function strikingly eradicated early B cell progenitor cells in vivo. Reduced survival fitness upon Lkb1 deletion in pre-B ALL cells was largely rescued by metabolites that can enter the TCA cycle and thus provide ATP. Importantly, we found that inducible deletion of Lkb1 delayed onset of pre-B ALL and prolonged survival of transplant recipient mice in vivo. Similar to observations made with deletion of Lkb1 in murine BCR-ABL1 pre-B ALL cells, both loss of Ampka function and small molecule inhibition of AMPK (BML-275) resulted in cell death as well as reduced glycolytic activity and mitochondrial function in BCR-ABL1 pre-B ALL cells. Moreover, prolonged overall survival was observed in transplant recipient mice injected with Ampka-deficient pre-B ALL cells. Finally, BML-275 synergized with glucocorticoids, a central component of all main therapy regimen in pre-B ALL, in eradicating patient-derived pre-B ALL cells. Conclusions: Taken together, our findings showed that B-cell lineage leukemia, unlike myeloid leukemia, critically depend on LKB1/AMPK signaling for survival. Our findings also showed that LKB1/AMPK can be leveraged to provide a novel therapeutic avenue in pre-B ALL. Disclosures Hochhaus: BMS: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; ARIAD: Honoraria, Research Funding.

Tumor Necrosis Factor-α Contributes to Microenvironmental up- Regulation of NF-κB Activity in Philadelphia Chromosome-Positive Acute Lymphoblastic Leukemia: Implication for a Novel Therapeutic Target.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1912-1912
Author(s):  
Hui-Jen Tsai ◽  
Seiichiro Kobayashi ◽  
Kiyoko Itoh ◽  
Takaomi Ishida ◽  
Kazuo Umezawa ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Therapeutic Target ◽  
In Vivo Imaging ◽  
Essential Role ◽  
Lymphoblastic Leukemia ◽  
Philadelphia Chromosome ◽  
Inhibitory Effect ◽  
Novel Therapeutic

Abstract Constitutively activated NF-κB has been demonstrated in primary blast cells and cell lines derived from Philadelphia chromosome (Ph- positive acute lymphoblastic leukemia(Ph- ALL). In our previous study, we have shown the essential role for NF-κB in growth and survival of Ph- ALL cells. To gain insight into the microenvironmental (cytokines and/or stromal cell) regulation of NF-κB activity in Ph- ALL, we lentivirally transduced Ph- ALL cells, IMS-PhL1 and Sup-B15 cells, with NF-κB/luciferase (kB/Luc) reporter construct and established a bioluminescence imaging model of Ph- ALL for in vitro and in vivo analysis. In in vitro study, we checked NF-κB/Luc activity by luminoter. It showed that NF-κB activity of Ph- ALL cells was significantly up-regulated by TNFa stimulation and synergistically augmented by seeding them onto a layer of murine HESS5 stromal cells, which singly didn’t change NF-κB activity in Ph- ALL cells. DHMEQ, a specific inhibitor of nuclear translocation of p65, eradicated constitutive and TNFa inducible NF-κB activity of Ph- ALL cells and induced their substantial apoptosis dose-dependently. However, the inhibitory effect of DHMEQ on TNFa induced NF-κB activity as well as viability of Ph- ALL was alleviated in the presence of HESS5. This alleviatory effect of DHMEQ induced NF-κB suppression by HESS5 was overcame by addition of TNFa inhibitor, Etanercept, in a dose of less than 1ug/ml. (Fig.1) Taken together, TNFa plays an essential role in up-regulation of NF-κB activity in the absence or presence of HESS5 cells. When Ph- ALL cells were treated with TPCA-1, an IKK-2 inhibitor, the TNFa induced NF-κB activity in Ph- ALL cells was suppressed even in the presence of HESS5 cells. Cell proliferation assay also showed inhibitory effect on proliferation of Ph- ALL cells by TPCA-1. In in vivo study, we transplanted Ph- ALL cells into NOD-Scid mice and periodically monitored the NF-κB activity of Ph- ALL cells by a CCD camera. Intriguingly, strong signal was detected in liver, stomach and ovary in addition to bone marrow, which was the predominant site of leukemic cell infiltration. QR-PCR analysis and immunohistochemistry staining for mouse tissue verified tumor infiltration up-regulate murine TNFa production in these tissues. It suggests the essential role of TNFa in the up-regulation of NF-κB signaling in mouse model. However high dose Etanercept, 1mg, subcutaneous injection into Ph- ALL cells transplanted mouse didn’t show significant reduction of NF-κB activity and partial response of NF-κB suppression was seen in the mouse injected with 5mg of Etanercept intraperitoneally. (Fig.2) This result suggests that factors other than TNFa may contribute to in vivo maintenance and/or up-regulation of NF-κB activity in Ph- ALL cells. In conclusion, TNFa-dependent and independent pathways are involved in microenvironmental up-regulation of NF-κB activity, which contribute to survival, expansion and presumably drug-resistance of Ph- ALL cells. The present bioimaging model helps us to dissect the regulatory mechanism of NF-κB signal in Ph- ALL and the results suggest us microenvironment as a novel therapeutic target in the treatment of Ph- ALL. Fig. 1 NF-κB activity of Sup-B15 cells treated with DHMEQ, TNFα and Etanercept(22hrs) Fig. 1. NF-κB activity of Sup-B15 cells treated with DHMEQ, TNFα and Etanercept(22hrs) Fig. 2 In vivo imaging of NF-κB activity in Sup-B15 cells treated with Etanercept Fig. 2. In vivo imaging of NF-κB activity in Sup-B15 cells treated with Etanercept

Functional Modulation of VLA6 in BCR-ABL1+ Pre-B Acute Lymphoblastic Leukemia.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2565-2565
Author(s):  
Eun Ji Gang ◽  
Yao-Te Hsieh ◽  
Huimin Geng ◽  
Jennifer Pham ◽  
Markus Muschen ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Drug Resistance ◽  
Acute Lymphoblastic Leukemia ◽  
Lymphoblastic Leukemia ◽  
Conditional Knockout ◽  
Leukemia Cells

Abstract Abstract 2565 Chemotherapy drug resistance in acute lymphoblastic leukemia (ALL) remains a major problem, resulting in reduced treatment efficacy and relapse. The bone marrow environment (BME) has been shown to promote resistance of leukemia cells towards chemotherapy, which has been attributed to several proteins, including integrins. Our analysis of 207 children with high-risk (BCR/ABL1−) pre-B ALL revealed that high expression of the laminin-binding integrin VLA6 (alpha6beta1) portends poor clinical outcomes in patients with minimal residual disease (MRD+) on day 29 of induction. In addition, our comparative analysis of pre-B leukemia and normal B-cells revealed that VLA6 is preferentially upregulated on BCR/ABL1+ pre-B ALL blasts. Alterations in adhesion properties have been described for BCR/ABL1+ (p210) chronic myeloid leukemia. The role of integrins and integrin VLA6 in particular for cell adhesion-mediated drug resistance (CAM-DR) in BCR/ABL1+ (p210) ALL has not been addressed. With respect to its role for normal immature hematopoietic cells, contradictory observations have been reported. Therefore, we hypothesized that VLA6-mediated adhesion of ALL cells to the bone marrow stromal niche contributes to drug resistance. We evaluated the role of VLA6 in BCR-ABL1+ leukemia using two of our established models of leukemia, a conditional knockout model of VLA6 in murine BCR-ABL1+ leukemia and a xenograft model of human BCR-ABL1+ leukemia. VLA6fl/fl cells were oncogenically transformed using BCR-ABL1 (p210) and cultured under lymphoid-skewing conditions. Induction of pre- B (B220+ CD19+) ALL was confirmed by flow cytometry. Subsequent transduction with CreERT2 or EmptyERT2 generated leukemia cells in which VLA6 ablation could be induced (CreERT2) or not (EmptyERT2) by addition of Tamoxifen. Conditional ablation of VLA6 in vitro decreased adhesion significantly compared to undeleted controls (19.7%±8.1% vs. 87.7%±6.0%; p=0.00041) and increased apoptosis of murine BCR-ABL1+ leukemia cells as determined by analysis of Annexin V−/7-AAD− viable cells by flow cytometry (VLA6 deleted vs. undeleted: 35.3%±1.1% vs. 75.1%±1.2%; p=0.0001). Moreover, VLA6 deletion sensitized murine ALL to a tyrosine kinase inhibitor (TKI), Nilotinib (p=0.022, 45.6%±2.4% vs. 73.3%±13.0%). To test the effect of VLA6 deletion on leukemic progression in vivo, VLA6 BCR/ABL1+ pre-B (B220+ CD19+) CreERT2+ or control transduced ALL cells were transferred into NOD/SCID mice. 3 days thereafter, VLA6 deletion was induced by Tamoxifen administration to all animals in 2 cycles for 5 days. In vivo deletion of VLA6 in delayed leukemia progression compared to VLA6 competent controls from a median survival time (MST) of 30 days post-leukemia injection to a MST of 43 days post-leukemia injection (p=0.008 Log-rank test). In vivo deletion of VLA6 in combination with Nilotinib treatment delayed leukemia progression compared to VLA6 competent, as Nilotinib-treated control animals have uniformly died of leukemia with a MST of 39.5 days, however the Nilotinib treated VLA6 deleted group is completely alive and is still being monitored (p=0.0025). When VLA6 was ablated before transfer and recipients were observed for leukemia progression, the recipients of VLA6–deficient murine leukemia cells also showed attenuated leukemia progression compared to recipients of VLA6 competent cells. Moreover, we show that VLA6 blockade de-adheres primary ALL cells from their cognate counter receptor laminin in vitro, and sensitizes primary ALL cells to TKI Taken together, modulating the function of VLA6 in ALL offers a new approach to overcome drug resistance in ALL. Given that VLA6 is probably largely redundant for normal immature hematopoiesis, this approach may be preferable over targeting of other integrins in BCR/ABL1+ leukemias on which VLA6 is expressed. Disclosures: No relevant conflicts of interest to declare.

Zebrafish Xenotransplantation and Focused Chemical Genomics: A Preclinical Therapeutic Model For T-Cell Acute Lymphoblastic Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2670-2670
Author(s):  
Victoria L Bentley ◽  
Chansey J Veinotte ◽  
Dale Corkery ◽  
Marissa A Leblanc ◽  
Karen Bedard ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Human Cancer ◽  
Lymphoblastic Leukemia ◽  
Zebrafish Embryos ◽  
Chemical Genomics ◽  
Cell Acute Lymphoblastic Leukemia

Abstract T-cell acute lymphoblastic leukemia (T-ALL) is a high-risk subset of ALL, for which there is a need for new therapeutic strategies and efficient preclinical screening methods. We have pioneered an innovative zebrafish human cancer xenotransplantation (XT) model to examine drug-tumor interactions in vivo. T-ALL cell lines and primary patient T-ALL samples were microinjected into 48-hour zebrafish embryos, a stage at which the adaptive immune system has not yet developed. Fluorescent labelling of tumor cells prior to injection and use of casper pigment mutant fish facilitates evaluation of drug response both by direct observation in transparent fish and enumeration of human cells following embryo dissociation. Proliferation rates are rapidly determined by directly counting fluorescent cells using in silico-based programs and/or utilizing immunohistochemical approaches to distinguish human cancer cells from host cell populations. T-ALL cell lines harboring defined mutations in the NOTCH1, phosphoinositide 3-kinase (PI3K)/AKT and mTOR pathways differentially responded to targeted inhibition using the γ-secretase inhibitor Compound E, triciribine, and rapamycin, when xenografted into embryos, consistent with responses in vitro. Primary patient-derived T-ALL bone marrow samples similarly engrafted and proliferated in zebrafish embryos. Using this in vivo chemical genomic approach, a targetable mutation sensitive to γ-secretase inhibition was identified from the diagnostic bone marrow sample of a child with T-ALL, which was confirmed by exome Sanger sequencing, and validated as a gain-of-function mutation in the NOTCH1 gene by luciferase assay and Western blot. Focused chemical genomics using the zebrafish T-ALL XT model provides a means of tailoring therapy using a real time in vivo assay that more accurately recapitulates the tumor microenvironment than in vitro methods and more rapidly than mouse xenografts. Moreover, the efficiency and cost-effectiveness of this innovative platform provides a novel intermediary for the prioritization of much-needed drug candidates in the preclinical pipeline. Disclosures: No relevant conflicts of interest to declare.

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