Cytochrome c–related caspase-3 activation determines treatment response and relapse in childhood precursor B-cell ALL

Blood ◽  
2006 ◽  
Vol 107 (11) ◽  
pp. 4524-4531 ◽  
Author(s):  
Lüder Hinrich Meyer ◽  
Leonid Karawajew ◽  
Martin Schrappe ◽  
Wolf-Dieter Ludwig ◽  
Klaus-Michael Debatin ◽  
...  
Keyword(s):  
Cytochrome C ◽  
B Cell ◽  
Caspase 3 ◽  
Single Cells ◽  
Lymphoblastic Leukemia ◽  
Disease Free Survival ◽  
Leukemia Cells ◽  
Cytochrome C Release ◽  
The Impact

AbstractDeficient activation of apoptosis signaling pathways may be responsible for treatment failure in acute leukemia. Here, we address the impact of intact apoptosis signaling in 78 patients with pediatric precursor B-cell acute lymphoblastic leukemia (ALL) by analysis of 2 key apoptogenic events: caspase-3 activation and cytochrome c release in leukemia cells cultured in vitro. Both events correlated only in the group of patients who had a good response and patients in continuous remission, suggesting that intact apoptosis signaling is a characteristic for favorable outcome. By combining both parameters, we identified a novel indicator, cytochrome c–related activation of caspase-3 (CRAC). CRAC directly connects the extent of caspase-3 activation to cytochrome c release in single cells in an individual patient sample. In CRAC-positive patients, indicating proficient apoptosis signaling, the number of persisting leukemia cells on day 15 was significantly lower than in the CRAC-negative patient group (n = 27, mean 6.0% versus n = 36, mean 22.6%; P = .003). At a median follow-up of 31 months, disease-free survival was 84 months (95% CI = 76 to 91 months) and 66 months (95% CI = 52 to 80 months) for patients with positive and negative CRAC, respectively (P = .019). CRAC may serve as a functionally defined risk factor for treatment stratification.

scholarly journals Comparative Studies of B Cell Precursor ALL Pathogenesis and Translational Biology Using a Conditional E2a-PBX1 Transgenic Mouse Model

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 283-283
Author(s):  
Jesus Duque-Afonso ◽  
Jue Feng ◽  
Florian Scherer ◽  
Zhong Wang ◽  
Michael L. Cleary
Keyword(s):  
B Cell ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cell ◽  
Disease Free Survival ◽  
Leukemia Cells ◽  
Mouse Strains ◽  
Driver Gene ◽  
Hematopoietic Stem

Abstract Pediatric acute lymphoblastic leukemia (ALL) represents a collection of orphan diseases that are defined by their genomic abnormalities. Their genetic diversity and low prevalence serve as major barriers to investigations of their molecular pathogenesis and translational biology. To address this, we have engineered novel mouse strains that conditionally activate and express E2a-PBX1, the fusion oncogene derived from chromosomal translocation t(1;19), present in 5-7% of pediatric ALL. Somatic activation of the oncogene is accomplished by Cre-recombinase expressed under the control of specific B-lineage promoters CD19 or Mb1, or in hematopoietic stem cells using the Mx1 interferon-inducible promoter. The three mouse strains show similar pre-leukemic and leukemic phenotypes. At the time of disease, mice exhibit leukocytosis, anemia and thrombocytopenia as well as lymphadenopathy and hepatosplenomegaly and infiltration of several organs including kidney, lung and central nervous system. Leukemia cell phenotypes (CD117+, CD19+, CD43+, CD45+, CD25+, sIgM-, Bp-1+, CD24+, CD127+, CD79a+ and TdT+) correspond to the phenotypic fraction B-C’ (or pro-B/large pre-B II stage, Basel nomenclature) that is very similar to human E2a-PBX1+ pre-B-ALL. Hence, we detected productive VDJ rearrangements and cytoplasmic heavy chain in 12.5 % of cases, a characteristic of human E2a-PBX1 leukemias. Leukemia incidence varies from 5-50% depending on the Cre driver gene and the median latency is about 10 months in E2a.PBX1/Mb1.Cre and Mx1.Cre lines, suggesting the need for secondary mutations. Whole exome sequencing detected secondary genetic aberrations, which were validated in a larger cohort of leukemias. Spontaneous deletions of Pax5, which are present in ~45% of pediatric ALLs with E2a-PBX1 gene fusions, were found in about 30 % of mouse E2a-PBX1 leukemias. Conditional deletion of Pax5 and E2a-PBX1 expression expanded progenitor B cell subpopulations in healthy 3-months old preleukemic mice. Consequently, Pax5 haplo-insufficiency in mice cooperates with E2a-PBX1 increasing the penetrance and shortening the latency of leukemia, providing the first evidence for cooperative oncogenic effects of Pax5 haplo-insufficiency. Tumor suppressor genes as Trp53 and Cdkn2a/b were inactivated by secondary mutations and deletions, respectively. Additionally, secondary recurrent activating mutations were detected in key signaling pathways such as Ras/Mapk and Jak/Stat on which the leukemia cells are strongly dependent. Furthermore, leukemia cells displayed higher basal levels of phosphorylated pSTAT5 and pAKT, pERK1/2, and were hyper-sensitive to stimulation with IL-7 and thymic stromal lymphopoietin (TSLP) as seen by induction of pSTAT5 and supported growth in colony-forming assays. The JAK1/2 inhibitor ruxolitinib blocked the induction of pSTAT5 by IL-7 and TSLP, inhibited colony formation in vitro, and increased disease-free survival after in vivo treatment. Human E2a-PBX1 primary cells and cell lines showed hypersensitivity to IL-7/pSTAT5 activation compared to other ALL karyotypes and pre-treatment with ruxolitinib blocked induction of pSTAT5 by IL-7. In summary, we have developed conditional transgenic E2a-PBX1 mouse models that consistently develop leukemias that resemble human pre-B-ALL carrying the t(1;19) and identified key cooperating oncogenic pathways. This model provides experimental validation of the multistep pathogenesis for a subset of ALL previously inferred from genomic analyses and provides a platform for comparative mechanistic and preclinical studies. Disclosures No relevant conflicts of interest to declare.

Intact apoptosis signaling in myeloid leukemia cells determines treatment outcome in childhood AML

Blood ◽  
2008 ◽  
Vol 111 (5) ◽  
pp. 2899-2903 ◽  
Author(s):  
Lüder H. Meyer ◽  
Manon Queudeville ◽  
Sarah M. Eckhoff ◽  
Ursula Creutzig ◽  
Dirk Reinhardt ◽  
...  
Keyword(s):  
Treatment Outcome ◽  
Cytochrome C ◽  
Myeloid Leukemia ◽  
Caspase 3 ◽  
Lymphoblastic Leukemia ◽  
Good Prognosis ◽  
Favorable Outcome ◽  
Leukemia Cells ◽  
Cytochrome C Release ◽  
Pediatric Aml

Recently we reported that intact apoptosis signaling is indicative of favorable outcome in childhood acute lymphoblastic leukemia. Here we addressed this issue in 45 pediatric acute myeloid leukemia patients analyzing 2 core apoptogenic events: cytochrome c release and caspase-3 activation. In patients with good prognosis cytochrome c release was clearly found to be caspasedependent and correlated with activated caspase-3, indicating that activation of initiator or amplifier caspases such as caspase-8 together with an intact apoptosome function are elementary for favorable outcome. The functional integrity of this apoptogenic checkpoint is reflected by the parameter caspase-dependent cytochrome c-related activation of caspase-3 (CRACdep). Patients with positive CRACdep values (intact signaling) exhibited superior survival compared with CRACdep negative patients (deficient signaling). Thus, the propensity to undergo apoptosis of leukemia cells is an important feature for favorable treatment outcome and may serve as an additional stratification tool for pediatric AML patients. This trial was registered at www.ClinicalTrials.gov as #NCT00111345.

JS-K, a Novel Nitric Oxide (NO) Generator, Induces Cytochrome c Release and Caspase Activation in HL-60 Myeloid Leukemia Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3415-3415
Author(s):  
Paul J. Shami ◽  
Vidya Udupi ◽  
Margaret Yu ◽  
Swati Malaviya ◽  
Joseph E. Saavedra ◽  
...  
Keyword(s):  
Cytochrome C ◽  
Caspase 3 ◽  
Myelogenous Leukemia ◽  
Leukemia Cells ◽  
Caspase 8 ◽  
Caspase 9 ◽  
Cytochrome C Release ◽  
Dose Dependent Fashion ◽  
Acute Myelogenous ◽  
Dose Dependent

Abstract NO induces differentiation and apoptosis in Acute Myelogenous Leukemia (AML) cells. Glutathione S-Transferases (GST) play an important role in multidrug resistance and are upregulated in 90% of AML cells. We have designed a novel prodrug class that releases NO on metabolism by GST. O2-(2,4-Dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate (JS-K, a member of this class) has potent antileukemic activity. We have previously shown that JS-K induces apoptosis in HL-60 cells by a caspase dependent mechanism (Molecular Cancer Therapeutics2:409-417,2003). The purpose of this study was to determine the pathway through which JS-K induces apoptosis. Western blot analysis showed that treatment of HL-60 cells with JS-K (0 – 1 μM) for 6 hours results in release of Cytochrome c from mitochondria in a dose dependent fashion. Treatment with JS-K resulted in a dose dependent activation of Caspase 9. Sixteen and 24 hours after exposure to 1 μM JS-K, Caspase 9 activity was induced by 393 ± 93% and 237 ± 13% of control, respectively (p = 0.03 at the 24 hours time point). Treatment with JS-K resulted in a dose dependent activation of Caspase 3. Twenty four hours after exposure to 1 μM JS-K, Caspase 3 activity was 208 ± 3.4 % of control (p = 0.02). Treatment with JS-K also resulted in a dose dependent activation of Caspase 8, but to a lesser extent than Caspase 9 and 3. Twenty four hours after exposure to 1 μM JS-K, Caspase 8 activity was 144 ± 5.3 % of control (p = 0.04). We conclude that JS-K activates the intrinsic pathway of apoptosis in leukemia cells by inducing the release of Cytochrome c from mitochondria. (NO1-CO-12400).

scholarly journals Obesity-Induced Chemoresistance in B-Cell Acute Lymphoblastic Leukemia Cells Is Mediated By Reduced Chemotherapy Uptake and Interleukin-9 Signaling

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 37-38
Author(s):  
Jamie A.G. Hamilton ◽  
Miyoung Lee ◽  
Ganesh R Talekar ◽  
Curtis J. Henry
Keyword(s):  
B Cell ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Pediatric Patients ◽  
Lymphoblastic Leukemia ◽  
Survival Pathways ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Interleukin 9 ◽  
The Impact

Background: Since the 1970's, obesity rates in children and adolescents have more than tripled (Ogden et al., 2006; Ogden et al., 2016) which parallels the rise in annual medical cost to treat obese pediatric patients in the United States which now exceeds $10 billion dollars (Beiner et al., 2020). The increasing cost to treat obese pediatric patients is in part due to more aggressive diseases which frequently present in these patients. For example, obese pediatric patients with B-cell acute lymphoblastic leukemia (B-ALL) have inferior overall survival compared to lean patients, with obese patients having a 2.5-fold increased risk of developing an adverse event and an almost 4-fold greater risk of death compared to non-obese patients (Butturini et al., 2007; Eissa et al., 2017; Ethier et al., 2012). Adipocytes accumulate with weight gain, and emerging data suggest the these cells secrete factors which promote chemoresistance in B-ALL cells (Ehsanipour et al., 2013; Sheng et al., 2016). Given the negative impact of obesity on cancer outcomes, we sought to define how adiposity impacts B-ALL pathogenesis and chemosensitivity. Methods: We performed global cytokine profiling and ELISA analyses to identify cytokines and chemokines secreted at high levels by adipocytes and present at high concentrations in the obese relative to the lean microenvironments. We then determined how candidate cytokines/chemokines impacted the function (proliferation/ signal transduction) of murine and human B-ALL cell lines using in vitro models of adiposity and the diet-induced murine model of obesity (DIO model). These experiments were also carried out on de-identified patient samples from lean and obese pediatric patients with B-ALL which were obtained from the Aflac Leukemia and Lymphoma Biorepository. To assess the impact of bone marrow stromal cells and adipocytes on B-ALL cell chemosensitivity, we exposed human B-ALL cells to stromal cell conditioned-media (SCM) and adipocyte-conditioned media (ACM) prior to methotrexate (MTX) treatment to determine their impact on cytotoxicity (Annexin-V/PI analysis) and drug uptake (confocal microscopy using fluorescently-conjugated methotrexate MTX). Results: We made the novel finding that Interleukin-9 is elevated in adipose-rich microenvironments and induces survival pathways which promote chemoresistance. From our studies, we found that obese mice succumb significantly faster than lean mice transplanted with murine Ph+ B-ALL cells. Analysis of serum obtained from lean and obese mice revealed that circulating IL-9 levels were significantly higher in mice with high adiposity. Furthermore, we found that adipocytes secreted copious amounts of IL-9; whereas, the production of this cytokine by bone marrow stromal cells was barely detectable. Interestingly, only human B-ALL cells exposed to ACM upregulated the Interleukin-9 receptor, suggesting increased sensitivity to this cytokine when malignant cells are exposed to adipocytes. Treating human B-ALL cells with recombinant IL-9 resulted in the activation of STAT3 and NF-κB signaling pathways and significantly increased proliferation. The induction of these proliferative and survival pathways coincided with reduced sensitivity to MTX treatment, whereas B-ALL cell death was abrogated when malignant cells were exposed to high levels of IL-9. Furthermore, the uptake of fluorescently-labeled MTX in ACM-exposed human B-ALL cells was significantly decreased compared to the internalization observed in B-ALL cells cultured in unconditioned media or SCM. Conclusions:It has been previously shown that IL-9 promotes chemoresistance in diffuse large B-cell lymphoma and is associated with worse prognosis in patients with B-cell chronic lymphocytic leukemia (Chen, Lv, Li, Lu, & Wang, 2014; Lv, Feng, Ge, Lu, & Wang, 2016). To our knowledge, our studies are the first to demonstrate that adipocyte-produced IL-9 promotes B-ALL pathogenesis and chemoresistance. To further explore this relationship and potential of targeting IL-9 as a novel therapeutic option, we are conducting in vitro and in vivo experiments to assess the impact of IL-9 neutralizing antibody treatment alone and when combined with frontline chemotherapy options for B-ALL Disclosures No relevant conflicts of interest to declare.

scholarly journals Resveratrol Targets Sphingolipid Metabolism and BCR-ABL in Ph+ Acute Lymphoblastic Leukemia to Induce Growth Inhibiton

Proceedings ◽  
2019 ◽  
Vol 40 (1) ◽  
pp. 3
Author(s):  
Oğuz ◽  
Adan
Keyword(s):  
Cell Cycle ◽  
Cell Growth ◽  
Cytochrome C ◽  
Protein Expression ◽  
Western Blot ◽  
Caspase 3 ◽  
Lymphoblastic Leukemia ◽  
Sphingolipid Metabolism ◽  
Parp Cleavage ◽  
Cytochrome C Release

The mechanisms underlying the growth inhibitory effect of resveratrol on Ph+ ALL cells were investigated with regard to targeting of ceramide metabolism and changes in BCR-ABL expression. Growth inhibition and apoptotic effects of resveratrol, SK inhibitor (SKI II), GCS inhibitor (PDMP), SPT inhibitor (myriocin) and resveratrol-inhibitor combinations were investigated by MTT cell proliferation test, Annexin-V/PI staining, caspase-3, PARP expression and cytochrome c release by western blot, while cytostatic effect was investigated by flow cytometry. The effect of resveratrol, inhibitors and combinations on BCR-ABL protein expression was determined by western blot. In addition, the effect of resveratrol on SPT, SK-1/2, GCS protein expression was determined by western blot. In both cell lines resveratrol and resveratrol with SKI II and PDMP suppressed cell growth, triggered apoptosis and arrested the cell cycle at S phase. The combination of resveratrol with myriocin showed cell-specific effects on cell growth and cell cycle, but triggered apoptosis in both cells. In both cell types, resveratrol and combinations generally increased cytochrome-c release, caspase-3 cleavage and PARP cleavage, but cell-specific changes were also detected. Resveratrol decreased the expression of SK-1/SK2 and GCS in both cells and increased SPT expression. While resveratrol, SKI II and PDMP decreased BCR-ABL expression and myriocin increased BCR-ABL expression. Resveratrol together with SKI II and PDMP caused increases in BCR-ABL, while combination with myriocin reduced BCR-ABL expression. As a result, resveratrol suppressed cell growth and triggered apoptosis in Ph+ ALL by regulating ceramide metabolism and BCR-ABL expression.

In-Vitro Treatment with the AKT-Inhibitor GSK 690693 Induces Cell Death in Lymphoma Cell Lines and in Primary CLL Cells and Is Followed by Caspase-3 Activation and Cytochrome C Release.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1589-1589
Author(s):  
Dirk Winkler ◽  
Thorsten Zenz ◽  
Daniel Mertens ◽  
Annett Habermann ◽  
Hartmut Döhner ◽  
...  
Keyword(s):  
Cell Death ◽  
Cytochrome C ◽  
Conditioned Medium ◽  
Cell Lines ◽  
Caspase 3 ◽  
Spontaneous Apoptosis ◽  
Akt Inhibitor ◽  
Cytochrome C Release ◽  
Viable Cells

Abstract The PI3K/AKT pathway acts as a critical regulator of cell survival by stimulating cell proliferation and inhibiting apoptosis and has been implicated in the pathogenesis of lymphoproliferative disorders. Therefore, inhibition of AKT seems to be a highly attractive new approach for the treatment of lymphoma. We treated 9 cell lines with AKT-nhibitor (1, 10, 20 μM) over 24h and 48h respectively: EHEB (B-CLL), GRANTA-519 (MCL), JURKAT (T-ALL) BL-60, NAMALWA and BJAB (all Burkitt’s lymphoma), L363, OPM-2 and RPMI-8226 (all multiple myeloma). To determine the rates and type of AKT-inhibitor induced cell death, FACS analyses for CD19, 7AAD, active caspase-3, cytochrome c were performed. The phosphorylation status of AKT and its downstream proteins GSK3β, p70S6k and S6 was studied by Western blotting after 5–120 minutes. In addition, 11 primary CLL samples with either del 13q (n=3), del 11q (n=2), del 17p (n=3) or a normal karyotype (n=3) were treated with AKT inhibitor (10 μM; 2.5μM; 0.625 μM; 0.156 μM). CLL samples were cultured in both standard medium as well as in HS5-(human stromal cells) conditioned medium to reduce spontaneous apoptosis of CLL in-vitro. 6 out of 11 patients had unmutated VH genes. 8 Patients were untreated, 3 were previously treated. Fludarabine (0.1 μM) was added to AKT-inhibitor in 11 cases to test for synergistic effects. CLL cells were harvested after 48 hours and 5 days to measure cell viability using Celltiter-GLO-Assay. Treatment of cell lines lead to significant rates of AKT-inhibitor induced cell death (table 1), to hyperphosphorylation of AKT and to inhibition of phosphorylation of GSK3β (after 5 min) and S6 (after 20 min) in all cell lines and of p70S6k (after 120 min) in GRANTA, JURKAT, NAMALWA and BJAB. Cell death did not depend on functional p53 gene. Treatment of primary CLL samples with AKT-inhibitor alone was followed by a decrease of cell viability in a time and concentration dependent manner regardless of the medium used (table 2). Only with the lowest concentration and when cultured in HS5-conditioned medium, no further reduction of viable cells was seen between 48h and 5d. Treatment with AKT-inhibitor as a single agent seemed to be at least as effective as treatment with fludarabine. Response was independent of the genetic subgroup, VH mutation status or prior treatment. High risk cases with del 17p responded worse to fludarabine alone when compared to cases without del 17p (i.e. 75% of viable cells after 5d at 10000 μM in cases with del 17p vs. 25% in cases without del 17p). The same fludarabine resistant cases showed good responses to treatment with AKT-inhibitor (9% of viable cells after 5d at 10000 μM in cases with del 17p). A synergistic effect was not achieved by combining AKT-inhibitor and fludarabine. Culture of CLL cells in HS5-conditioned medium resulted in lower rates of spontaneous apoptosis, but also in lower rates of AKT-inhibitor induced cell death. In conclusion, in-vitro treatment with AKT-inhibitor resulted in significant rates of cell death in cell lines and primary CLL cells, even in patients with del 17p or resistance to fludarabine. In cell lines, treatment with AKT-inhibitor was followed by typical features of apoptosis such as activation of caspase-3 and cytochrome c release. In CLL samples, prior treatment did not affect in-vitro response rates. These data underline the involvement of the PI3K/Akt pathway in the pathogenesis of lymphoma and point to an efficacy of the AKT-inhibitor in the treatment of lymphoma, multiple myeloma and CLL in-vivo. Concerning CLL, the AKT-inhibitor seems to be an attractive new treatment option even for cases with high risk cytogenetics. Using HS5-conditioned medium seems to be a well functioning method to reduce spontaneous apoptosis of CLL cells in-vitro. Table 1: rates of cell death, caspase-3 activation and cytochrome c release after treatment of cell lines with AKT inhibitor (1μM, 48h) 7AAD-positive cells active caspase-3 cytochrome c release EHEB 15% − + GRANTA-519 15% + + JURKAT 17% + + BL60 24% + + NAMALWA 25% − (+) BJAB 30% + (+) L363 15% + − OPM-2 41% + + RPMI-8226 32% + (+) Table 2: mean percentage of viable cells after treatment with AKT-Inhibitor (A), fludarabine (F; 0,1μM) and their combination (A + F) measured by Celltiter-GLO-Assay 10000 nM 2500 nM 625 nM 156,25 nM 48h 5d 48h 5d 48h 5d 48h 5d A F A + F A F A + F A A F A+ F A F A+ F A HS5 + (n=8) 94 84 (n=5) 75 (n=5) 45 22 (n=5) 25 (n=5) 88 52 91 84 80 69 22 18 76 85 HS5 − (n=11) 60 79 59 8 39 21 77 27 80 79 76 28 39 34 82 (n=10) 21

Obinutuzumab (GA101) Significantly Enhances Cell Death and ADCC Compared to Rituximab Against CD20+ sensitive and Rituximab Resistant B-Cell Non-Hodgkin Lymphoma (NHL) and Lymphoblastic Leukemia (BLL)

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4865-4865 ◽  
Author(s):  
Aradhana Awasthi Tiwari ◽  
Janet Ayello ◽  
Carmella van de Ven ◽  
Danielle Glassman ◽  
Anthony Sabulski ◽  
...  
Keyword(s):  
Cell Death ◽  
B Cell ◽  
Nk Cells ◽  
Cell Lines ◽  
Caspase 3 ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Dismal Prognosis

Abstract Abstract 4865 Background: Patients who relapse with CD20+ B-NHL and B cell lymphoblastic leukemia (B-LL) have a dismal prognosis, often associated with chemotherapy resistance (Cairo et al. JCO, 2012,Mils/Cairo et al. BJH,2012) and often require alternative therapeutic strategies. Rituximab (RTX) in combination with FAB 96 chemotherapy is a safe, well-tolerated treatment that is associated with > 90% EFS in children with newly diagnosed and advanced mature B-Cell NHL (Cairo M.S. et al. ASCO, 2010). Resistance to RTX, however, may predispose patients with CD20+ NHL to an increase risk of relapse and or disease progression (Barth/Cairo et al. BJH, 2012; Tsai et al. Cl. Can. Res, 2012). Obinutuzumab (GA101), a novel type II glycoengineered CD20 antibody of the IgG1 isotype, mediates enhanced cell death vs RTX and has a glycoengineered Fc region that induces significantly enhanced ADCC (Mössner et al. Bld, 2010; Niederfellner G. et al. Bld, 2011; Bologna L et al. JI, 2012). Objective: To evaluate the in-vitro efficacy of GA101 compared to RTX against RTX sensitive and resistant CD20+ B-NHL and B-LL cell lines. Methods: Raji (CD20+,ATCC, Manhass, VA), U698-M (CD20+, DSMZ, Germany), Loucy cells (CD20−) (T-ALL) (ATCC, Manhass, VA) and Raji-2R and Raji-4RH (generously supplied by M. Barth, Roswell Park Cancer Institute) were cultured in RPMI with 10% FBS and incubated with GA101 and/or RTX at 100 μg/ml for 24 hrs (n=6), 48 and 72 hrs (n=5). Cell death was evaluated by staining with AnnexinV/7AAD and flow-cytometry. Loucy cells (CD20−) were used as the negative control. The caspase 3/7 activity was measured by FAM caspase 3/7 assay kit by FLICA™ methodology. RSCL, RRCL, U698-M and Loucy were incubated with GA101 and RTX treatment for 24, 48 and 72 hrs, and caspase3/7 activity was detected by FACS using 488 nm excitation and emission filter (n=3). ADCC were performed with K562-IL-15–41BBL expanded NK cells (Ayello/Cairo et al. ASH, 2010) as well as IL-2 expanded NK cells, at 20:1 effector: target ratio (E: T, n=3) using europium release assay (Perkin-Elmer). Results: GA101 induced significantly more cell death compared to RTX in B-NHL and BLL cell lines. (Table-1) GA101 vs RTX shows a significantly increase in caspase 3/7 activity in Raji 16.92±0.84% vs 11.76±0.08% compared to Raji2R 6.7±0.62% vs 2.8±0.7%, Raji4RH 5.8±0.35% vs 2.0±0.3% and U698-M 12.54±0.44% vs 9.6±0.95% compared to Loucy 3.22±0.45% vs 2.59±0.05%, respectively, at 24 hrs of treatment (p<0.0001). GA101 vs RTX also elicited a significant increase a ADCC with K562-IL15–41BBL expanded NK cells, in Raji 73.8±8.1% vs 56.81±4.6% compared to Raji-2R 38.0±2.0% vs 21.6±1.2%, Raji-4RH 40.0±1.6% vs 0.5±1.1% and U698-M 70.0±1.6% vs 45.56±0.1%, compared to Loucy 21.67±0.48% vs 15.92±0.52%, respectively (p<0.001) at day 7.The IL-2 alone expanded Hu-NK cells demonstrated a reduction of 10–20% cytotoxicity compared to K562-IL15–41BBL Hu-NK cells at day 7 against BLL, RSCL and RRCL, in-vitro. Conclusion: Obinutuzumab compared to RTX significantly enhanced cell death, caspase3/7 activity and NK mediated ADCC in sensitive and RTX resistant B-NHL and B-LL. Obinutuzumab represents a promising candidate for treating RTX sensitive and resistant CD20+ B-Cell Lymphomas and lymphoblastic leukemia. Further studies will investigate the combination of activated NK cells or chemotherapy that may enhance or synergize with the efficacy of GA101 (Obinutuzumab) both in -vitro and in-vivo in xenografted NOD/SCID mice. Disclosures: No relevant conflicts of interest to declare.

scholarly journals CD20 up-regulation in pediatric B-cell precursor acute lymphoblastic leukemia during induction treatment: setting the stage for anti-CD20 directed immunotherapy

Blood ◽  
2008 ◽  
Vol 112 (10) ◽  
pp. 3982-3988 ◽  
Author(s):  
Michael N. Dworzak ◽  
Angela Schumich ◽  
Dieter Printz ◽  
Ulrike Pötschger ◽  
Zvenyslava Husak ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
B Cell ◽  
Residual Disease ◽  
Lymphoblastic Leukemia ◽  
Leukemic Cells ◽  
Induction Treatment ◽  
Cell Precursor ◽  
Anti Cd20 ◽  
The Impact

Abstract CD20 is expressed in approximately one- half of pediatric acute lymphoblastic leukemia (ALL) cases with B-cell precursor (BCP) origin. We observed that it is occasionally up-regulated during treatment. To understand the impact of this on the potential effectiveness of anti-CD20 immunotherapy, we studied 237 CD10+ pediatric BCP-ALL patients with Berlin-Frankfurt-Munster (BFM)–type therapy. We analyzed CD20 expression changes from diagnosis to end-induction, focusing on sample pairs with more than or equal to 0.1% residual leukemic blasts, and assessed complement-induced cytotoxicity by CD20-targeting with rituximab in vitro. CD20-positivity significantly increased from 45% in initial samples to 81% at end-induction (day 15, 71%). The levels of expression also increased; 52% of cases at end-induction had at least 90% CD20pos leukemic cells, as opposed to 5% at diagnosis (day 15, 20%). CD20 up-regulation was frequent in high-risk patients, patients with high minimal residual disease at end-induction, and patients who suffered later from relapse, but not in TEL/AML1 cases. Notably, up-regulation occurred in viable cells sustaining chemotherapy. In vitro, CD20 up-regulation significantly enhanced rituximab cytotoxicity and could be elicited on prednisolone incubation. In conclusion, CD20 up-regulation is frequently induced in BCP-ALL during induction, and this translates into an acquired state of higher sensitivity to rituximab. This study was registered at http://www.clinicaltrials.gov as #NCT00430118.

Novel Targeted Therapy for Precursor B Cell Acute Lymphoblastic Leukemia: Anti-CD22 Antibody-MXD3 Antisense Oligonucleotide Conjugates

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3719-3719
Author(s):  
Noriko Satake ◽  
Connie Duong ◽  
Michael Oestergaard ◽  
Sakiko Yoshida ◽  
Cathy Chen ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
B Cells ◽  
B Cell ◽  
Antisense Oligonucleotide ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cells ◽  
High Dose ◽  
Treatment Groups ◽  
Primary Leukemia

Abstract Precursor B cell (preB) acute lymphoblastic leukemia (ALL) is the most common cancer in children. Despite aggressive treatment, current approaches for preB ALL have significant limitations with a cure rate of only 30% in certain subtypes. In addition, long-term survivors are at risk for late effects that include development of secondary malignancies and toxicity to visceral organs. Therefore, there is a desperate need for more effective and less toxic therapy. Treatments and outcome of the animals. Dash-dots: PBS, dots: MXD3 ASO and aCD22 Ab unconjugated, gray solid: aCD22 Ab-ASO conjugates at 0.2mg of Ab/kg/dose, and black solid: aCD22 Ab-ASO conjugates at 1mg of Ab/kg/dose Treatments and outcome of the animals. Dash-dots: PBS, dots: MXD3 ASO and aCD22 Ab unconjugated, gray solid: aCD22 Ab-ASO conjugates at 0.2mg of Ab/kg/dose, and black solid: aCD22 Ab-ASO conjugates at 1mg of Ab/kg/dose Previously, we demonstrated that the transcription factor MXD3 is a critical regulator of preB ALL cell proliferation. Knockdown of MXD3 expression in preB ALL cells resulted in cell death as determined by annexin V and caspase assays (Satake et al., British Journal of Haematology, in press). We hypothesize that targeted delivery of an MXD3 therapeutic to preB ALL cells will increase therapy effectiveness and decrease off target effects and toxicity, thus increasing the quality of life. In the current study, we developed an antisense oligonucleotide (ASO) that specifically targets MXD3. Taking advantage of anti-CD22 antibody (aCD22 Ab), which specifically targets B cell ALL, we developed aCD22 Ab-MXD3 ASO conjugates as a potential combined phenotypic and molecularly targeted therapeutic. In in vitro tests, using the Reh ALL cell line, we confirmed successful delivery of the aCD22 Ab-MXD3 ASO compound to leukemia cells. This correlated with MXD3 knockdown at the protein level, and inhibition of leukemia cell growth. Cytotoxicity was tested on anonymized healthy donor normal blood cells, including CD34 positive hematopoietic stem cells (HSCs), B cells, and non-B cells from mobilized peripheral blood mononuclear cells. As expected, cytotoxicity was observed in normal B cells, but CD34 HSCs and non-B cells were unaffected. In combination with conventional preB ALL chemotherapy drugs (vincristine or doxorubin), we observed additive cytotoxic effects of aCD22 Ab-MXD3 ASO conjugates on cells in vitro. We determined the compound effectiveness in pre-clinical xenograft animal models of preB ALL, using both the Reh cell line as well as a primary leukemia sample. Age matched female NOD/SCID/IL2Rg-/- (NSG) mice were randomly assigned to 4 treatment groups: 1) PBS, 2) MXD3 ASO and aCD22 Ab unconjugated at the equivalent dose of the high dose group, 3) aCD22 Ab-ASO conjugates at 0.2mg of Ab/kg/dose, and 4) at 1mg of Ab/kg/dose. Five million leukemia cells were inoculated either intravenously (iv) (Reh) or intra-bone marrow (primary cells) to each mouse. Twenty-four hours following leukemia inoculation, animals started receiving twice weekly iv treatments for 3 weeks. All mice treated with PBS and unconjugated MXD3 ASO & aCD22 Ab, died of leukemia at approximately day 21 in the Reh model and at day 30 in the primary leukemia model (Figure). Animals treated with aCD22 Ab-MXD3 ASO conjugates, either at low or high dose, had significantly prolonged survival both in the Reh (p<0.0084, n=4/group) and primary leukemia (p=0.0001, n=8/group) xenograft models (Log-rank (Mantel-Cox) test). Leukemia-related death was confirmed by necropsy. Harvested leukemia cells were HLA and CD22 positive. During treatment, the mice in all the treatment groups remained healthy and active, and did not lose weight. Toxicity was assessed in the primary leukemia model with weekly CBC and chemistry panels and revealed no significant toxicity. In conclusion, we have demonstrated the therapeutic efficacy of the aCD22 Ab-MXD3 ASO conjugates in preB ALL. This is the first study to demonstrate effective direct ASO-conjugated monoclonal Ab-mediated delivery for the treatment of leukemia or other malignancies. Future studies will focus on the further characterization of the compound, effectiveness of combination therapy with standard chemotherapeutic drugs and toxicological profile. Disclosures No relevant conflicts of interest to declare.

Sign in / Sign up

Export Citation Format

Share Document