Anti-Leukemic Activity of a Novel Pegylated Recombinant Erwinia Chrysanthemi-Derived L-Asparaginase On Lymphoid Cell Lines and Leukemia-Bearing Mouse Models.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2571-2571 ◽  
Author(s):  
Wei-Wen Chien ◽  
Céline Lebeux ◽  
Nicolas Rachinel ◽  
Soraya Allas ◽  
Pierre Sahakian ◽  
...  
Keyword(s):  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Lymphoma Cell ◽  
Erwinia Chrysanthemi ◽  
Leukemic Cells ◽  
Hypersensitivity Reactions ◽  
Sensitive Cell ◽  
Development Fund

Abstract Abstract 2571 Background: Bacterial-derived L-asparaginase (ASNase) is an indispensable component of the therapy of acute lymphoblastic leukemia (ALL). Despite the high rate of successful treatment, hypersensitivity reactions occur in 20–40 % of patients receiving this non-human protein, which limits the use of ASNase. Native Erwinia chrysanthemi-derived ASNase (n-crisantaspase) has been used for treating patients exhibiting allergic symptoms to native and PEGylated Escherichia coli-derived ASNases (EC-ASNase). However, hypersensitivity reactions are still observed in at least 17 % of patients receiving n-crisantaspase. A PEGylated recombinant Erwinia chrysanthemi-derived ASNase (PEG-r-crisantaspase) with improved pharmacokinetic and pharmacodynamics properties and reduced immunogenicity has been developed recently (Allas et al., abstracts #2003, #2034, ASH 2009). We present here the in vitro and in vivo evaluation of PEG-r-crisantaspase on leukemia and lymphoma cell lines and on a leukemia-bearing mouse model, respectively. Material and methods: Different cell lines (ALL, B, T and NK/T lymphoma) and bone marrow aspiration samples obtained from patients with B-ALL or T-ALL were exposed in vitro to increasing doses (0.00005 to 5 U/ml) of PEG-r-crisantaspase, n-crisantaspase, or native EC-ASNase for 3 days. The cytotoxicity of each molecule was evaluated using 3- (4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) test. For the in vivo study, 5 millions of ASNase-sensitive cells, RS(4,11) were xenografted by intravenous injection (iv) into immunodeficient NOD/SCID mice. When 5% of leukemic blasts were observed in the blood of leukemia-bearing mice, 2, 5, 10 or 20 U/kg of PEG-r-crisantaspase or vehicle was injected intravenously in those mice. Two additional injections were performed with an interval of 7 days. Mice having significant weight loss (>20%) and/or extensive expansion of leukemic cells in blood (> 25%) and/or impaired general condition were sacrificed. Results: In vitro, PEG-r-crisantaspase and n-crisantaspase exhibited similar half maximal effective concentration (EC50) values for inhibiting the proliferation of leukemia and lymphoma cells. PEG-r-crisantaspase had a greater cytotoxicity effect on two high-sensitive cell lines than n-crisantaspase did, as shown by 4.5 and 8.1-fold-weaker EC50 values of PEG-r-crisantaspase relative to n-crisantaspase. PEG-r-crisantaspase, r-crisantaspase and n-crisantaspase were more efficient than EC-ASNase on less sensitive cell lines, which might be related to the 10-fold-greater glutaminase activity of crisantaspases than EC-ASNase. In vivo, after the first administration of PEG-r-crisantaspase in animals with ≥ 5% of leukemic cells, the leukemic cells were reduced to almost 0 % in mice within 4 days with all doses of PEG-r-crisantaspase tested, whereas the leukemic cells kept multiplying in the mice receiving vehicle. The 3 repeated injections of PEG-r-crisantaspase with an interval of 7 days delayed leukemia development for 17 days. All control mice receiving the vehicle had to be sacrificed at day 44 after leukemia inoculation, whereas of 28 mice receiving PEG-r-crisantaspase, 5 reached this endpoint at day 51 and the others at day 56. PEG-r-crisantaspase significantly improved the survival of leukemia-bearing mice for 7 to 12 days, regardless of the dose tested. Conclusions: These data provide evidence that PEG-r-crisantaspase has similar in vitro cytotoxic effect to n-crisantaspase on leukemia and lymphoma cell lines and significantly reduces the expansion of leukemic cells in leukemia-bearing mice, prolonging the survival of the animals. These results, together with preclinical PK/PD and immunogenicity data, support the clinical development of PEG-r-crisantaspase. A phase I dose escalation study in adult patients with relapsed or refractory hematological malignancies has been recently initiated. Research support from Alizé Pharma, the European Regional Development Fund (ERDF) and Grand Lyon. Disclosures: Allas: Alizé pharma: Employment. Sahakian:Alizé pharma: Employment. Julien:Alizé pharma: Employment. Abribat:Alizé pharma: Employment.

Variability In Precursor B ALL Killing With Moxetumomab Pasudotox (CAT-8015) Linked To Differential Binding and Endocytic Trafficking

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 5022-5022
Author(s):  
Ksenia Matlawska-Wasowska ◽  
Christian K. Nickl ◽  
Stuart S. Winter ◽  
Patricia Burke ◽  
Theresa LaVallee ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Response To Treatment ◽  
In Vivo Studies ◽  
Sensitive Cell ◽  
Work Related ◽  
Comprehensive Picture

Abstract Precursor-B acute lymphoblastic leukemia (pre-B ALL) is the most common malignancy in children and can be cured in a majority of patients. However, cure remains elusive in approximately 20% of patients for reasons that are not well understood. Moxetumomab pasudotox is one of several CD22-targeting therapies currently under investigation in clinical trials for subjects with B-cell malignancies, including pediatric subjects with pre-B ALL. Moxetumomab pasudotox (MP) is a second-generation immunotoxin composed of disulfide-stabilized, affinity matured VL and VH regions of the mouse anti CD22 monoclonal antibody RFB4 fused to PE38, a truncated form of Pseudomonas exotoxin E. We evaluated in vitro activity of MP against six pre-B ALL cell lines (697, Nalm6, MHH-Call3, RS4;11, SupB15, REH), as well as freshly isolated patient blasts. We found the most sensitive cell line to be Reh, followed by 697, MHH-Call3, and the least sensitive cell lines to be Nalm6 and RS4;11. Toxicity of MP on ALL cell lines and patient blasts was not well-correlated with the number of the CD22 receptors present on the cell surface. However, we found that cleavage of MP by ALL cell lines into active toxin correlated with uptake and inhibition of protein synthesis. Using ALL cell lines, we also demonstrated that binding and internalization of MP/CD22 complexes was correlated with pre-B ALL cell line responses to MP. In addition, the Fv MP/CD22 complexes internalized more slowly than the parent RFB4 antibody/CD22 complexes. In addition to evaluating cell lines, we applied similar assays to 7 patient samples of bone marrow blasts, where we evaluated bound and internalized MP with cellular toxicity up to 72 hours. Our results suggest that some of the heterogeneity observed in in vitro responses to treatment with MP may be related to differences in internalization. Additional studies evaluating intracellular cleavage and trafficking/processing of MP in cell lines and patient blasts is ongoing. These studies, combined with planned in vivo studies evaluating MP antitumor efficacy using ALL cell lines and patient blasts, will provide a more comprehensive picture for differences in ALL response to treatment with MP. TLaVallee currently affiliated with Kolltan Pharmaceuticals, New Haven, CT, USA; work related to this abstract occurred at MedImmune. Disclosures: Burke: MedImmune: Employment. LaVallee:MedImmune: full employee when work was conducted Other.

In vitro cytotoxicity of docetaxel in childhood acute leukemias.

1998 ◽  
Vol 16 (3) ◽  
pp. 907-913 ◽  
Author(s):  
R Consolini ◽  
C H Pui ◽  
F G Behm ◽  
S C Raimondi ◽  
D Campana
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Philadelphia Chromosome ◽  
Leukemic Cells ◽  
Allogeneic Bone ◽  
Acute Leukemias ◽  
Growing Cell

PURPOSE In seeking to identify novel effective antileukemic agents, we assessed the in vitro activity of the taxoid docetaxel (Taxotere; Rhone-Poulenc Rorer, Antony, France) in primary leukemic cells supported in culture by bone marrow-derived stromal layers. MATERIALS AND METHODS Bone marrow samples from children with acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) were cultured on allogeneic bone marrow-derived stromal layers and exposed to various concentrations of docetaxel. After 7 days of culture, the number of viable leukemic cells were counted by flow cytometry and compared with that in parallel cultures without drugs. RESULTS In 20 samples tested (15 B-lineage ALL, one T-lineage ALL, and four AML), the median cytotoxicity was 78% after a 7-day culture in the presence of 100 ng/mL docetaxel (range, 54% to 95%). The effects were dose-dependent and extended to all five ALL samples with the t(9;22)(q34;q11) (Philadelphia chromosome) or 11q23 abnormalities, karyotypes associated with an unfavorable outcome. Studies with continuously growing cell lines demonstrated that docetaxel exerted its cytotoxic effect by inducing apoptosis, and was consistently more effective than paclitaxel (Taxol; Bristol-Myers Squibb, Wallingford, CT) (mean 50% cell kill [LC50], 6.93 v 12.86 ng/mL in six leukemic cell lines). The antileukemic activities of docetaxel and vincristine were synergistic. While the mean (+/- SD) cytotoxicity of vincristine (0.1 ng/mL) was 11.2% +/- 7.3% and that of docetaxel (10 ng/mL) was 19.3% +/- 17.5% in CEM-C7 cells after 24 hours, combining the two agents increased the cytotoxicity to 62.5% +/- 20.7% (P = .003). CONCLUSION Docetaxel, at concentrations achievable in vivo, is cytotoxic to ALL and AML cells. These results provide a rationale for clinical trials of docetaxel in patients with acute leukemia.

Preclinical Activity of LBH589 Alone or in Combination with Chemotherapy in a Xenogeneic Mouse Model of Human Acute Lymphoblastic Leukemia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1520-1520
Author(s):  
Xabier Agirre ◽  
Amaia Vilas-Zornoza ◽  
Gloria Abizanda ◽  
Cristina Moreno ◽  
Victor Segura ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Acute Lymphoblastic Leukemia ◽  
Mouse Model ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Leukemic Cells ◽  
Regulatory Function ◽  
Human Leukemia

Abstract Abstract 1520 Histone deacetylases (HDACs) have been identified as therapeutic targets due to their regulatory function in chromatin structure and organization. Here we analyzed the therapeutic effect of LBH589 or panobinostat, a class I-II HDAC inhibitor, in acute lymphoblastic leukemia (ALL). In vitro, LBH589 induced a significant dose-dependent increase in cell apoptosis and a markedly inhibition of cell proliferation, which were associated with increased H3 and H4 histone acetylation. While apoptosis of ALL cells was detected between 12 and 24 hours after treatment with LBH589, changes in acetylated H3 and H4 were detected as early as 2 hours. Phosphorylation of H2AX, as an early marker of DNA damaged, was detected 12 to 24 hours after in vitro treatment with LBH589. These results suggest that H3 and H4 acetylation precede DNA damaged and induction of apoptosis indicating that inhibition of HDAC is likely to be responsible at least in part for LBH589 induced apoptosis and inhibition of cell proliferation. The in vivo activity of LBH589 was initially examined in a subcutaneous ALL mouse model. The ALL cell lines TOM-1 and MOLT-4 were transplanted (1×106 cell per animal) subcutaneously into the left flanks of 6-week-old female BALB/cA-Rag2−/−γc−/−. These cell lines develop into a rapidly growing tumor. Treatment with 5mg/kg of LBH589 was initiated 24 hours after injection of the leukemic cells, included 3 cycles of 5 consecutive days of LBH589 with two days rest between cycles and animals were monitored for 24 days. A significant inhibition of tumor growth was demonstrated in animals treated with LBH589 compared with control animals (P <0.01). Inhibition of leukemia cell growth was associated with an increase in the levels of acetylated H3 and H4 and an increase in phosphorylated H2AX in the leukemic cells obtained after sacrifice of mice. These results suggest that LBH589 has a powerful antileukemic effect not only in vitro but also in vivo. Using primary ALL cells, a xenograft model of human leukemia in BALB/c-RAG2−/−γc−/− mice was established, allowing continuous passages of transplanted cells to several mouse generations. A total of 10 million cells from a patient with T-ALL (ALL-T1) and a patient with B-ALL (ALL-B1) were administered intravenously into the tail vein of 6-week-old immunodeficient female BALB/cA-Rag2−/−γc−/− mice. Kinetics of engraftment of leukemic cells was monitored in PB and BM by phenotyping while organ infiltration was analyzed by immunohistochemistry. There were no significant differences in the genome, methylome or transcriptome between the original sample and the samples obtained after multiple generations on mice. To determine the efficacy of LBH589 alone or in combination with drugs currently used for treatment of ALL, BALB/cA-RAG2−/−γc−/− mice engrafted with ALL-T1 and ALL-B1 cells were treated with LBH589, Vincristine and Dexamethasone or a combination of LBH589 with Vincristine and Dexamethasone. Treatment was initiated when disease could be detected in PB by FACS (24 hours after injection of cells for ALL-T1 and between day 17 and 21 after injection for ALL-B1). LBH589 was administered i.p. on days 1–5, 8–12 and 15–19, Vincristine i.v. on days 1, 8 and 21 and Dexamethasone daily until day 21 i.p. and survival was analyzed. Treatment of mice engrafted with T or B-ALL cells with LBH589 induced an in vivo increase in the acetylation of H3 and H4, which was accompanied with prolonged survival of LBH589-treated mice in comparison with those receiving Vincristine and Dexametasone. Notably, the therapeutic efficacy of LBH589 was significantly enhanced in combination with Vincristine and Dexametasone. Our results demonstrate the therapeutic activity of LBH589 in combination with standard chemotherapy in pre-clinical models of ALL and suggest that this combination may be of clinical value in the treatment of patients with ALL. Disclosures: No relevant conflicts of interest to declare.

Serial Transplantation of a Human Acute T Lymphoblastic Leukemia into Nude Mice

1986 ◽  
Vol 72 (6) ◽  
pp. 553-558 ◽  
Author(s):  
Maria Giovanna Martinotti ◽  
Roberto Arione ◽  
Roberto Foà ◽  
Luigi Pegoraro ◽  
Cristina Jemma ◽  
...  
Keyword(s):  
Nude Mice ◽  
Lymphoblastic Leukemia ◽  
Experimental System ◽  
Leukemic Cells ◽  
Immune Reactivity ◽  
Chromosomal Markers ◽  
Total Body ◽  
Serial Transplantation

A human acute T lymphoblastic leukemia line (PF-382) was serially transplanted into nude mice. No takes were observed in untreated nude mice, whereas solid tumors were observed in splenectomized and total body, sublethally irradiated mice. The minimal tumor-inducing dose and the latency time remained unchanged after the third and fifth serial transplants. Moreover, leukemic cells recovered from the 8th in vivo passages displayed the same differentiation antigens and chromosomal markers as the in vitro PF-382 cell line used for the first transplant. This stable and well-characterized experimental system could be a new model for T-lymphocyte differentiation and immune-reactivity against human leukemias.

EB10, an Anti-FLT3 Monoclonal Antibody, Selectively Targets ALL Cell Lines and Primary ALL Blasts without Interfering with Normal Hematopoiesis.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2744-2744 ◽  
Author(s):  
Obdulio Piloto ◽  
Patrick Brown ◽  
Li Li ◽  
Bao Nguyen ◽  
Kyu-Tae Kim ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Cell Lines ◽  
Scid Mice ◽  
Cytotoxic Agents ◽  
Radioactive Isotopes ◽  
Leukemic Cells ◽  
Class Iii ◽  
Antibody Treatment

Abstract The class III receptor tyrosine kinase, FLT3, is expressed by &gt;90% of B-lineage acute lymphoblastic leukemias (ALL) blasts. In addition, it is expressed at extremely high levels in ALL patients with MLL-rearrangements or hyperdiploidy and sometimes mutated in these same patients. In this report, we investigated the effects of EB10, an anti-human FLT3 monoclonal antibody capable of preventing binding of FLT3 ligand (FL), on ALL cell lines and primary cells. In vitro studies, examining the ability of EB10 to inhibit FLT3 activation and downstream signaling in ALL cell lines and primary blasts, yielded variable results. In some cell lines FLT3 phosphorylation was inhibited and with it, downstream activation of pathways involving MAPK, AKT, and STAT5 phosphorylation. However, several cell lines actually exhibited FLT3 activation upon antibody treatment, possibly because of antibody-mediated receptor dimerization, and subsequent activation of downstream pathways. Nevertheless, through antibody-mediated cellular cytotoxicity (ADCC) such an antibody could still prove efficacious against leukemia cells in vivo. In fact, EB10 treatment significantly prolongs survival and/or reduces engraftment of ALL cell lines and primary ALL blasts in NOD/SCID mice. This effect might be even more pronounced in a host that was less immune compromised than are NOD/SCID mice. The leukemic cells surviving EB10 treatment in the mice were characterized by FACS analysis and found to express low levels or no FLT3. In contrast to the reduction in engraftment of human ALL primary blasts, EB10 treatment of NOD/SCID mice did not reduce engraftment of human hematopoietic CD34+ cells. Taken together, these data demonstrate that EB10 is selectively cytotoxic to ALL blasts while having little effect on normal hematopoiesis. Such an antibody, either naked or conjugated to radioactive isotopes or cytotoxic agents, may prove useful in the therapy of infant ALL as well as childhood and adult ALL patients whose blasts typically express FLT3.

IPI-504, a Novel, Orally Active HSP90 Inhibitor, Prolongs Survival of Mice with BCR-ABL T315I CML and B-ALL.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2183-2183 ◽  
Author(s):  
Cong Peng ◽  
Julia Brain ◽  
Yiguo Hu ◽  
Linghong Kong ◽  
David Grayzel ◽  
...  
Keyword(s):  
Cell Lines ◽  
Median Survival ◽  
Combination Treatment ◽  
Lymphoblastic Leukemia ◽  
Hsp90 Inhibitor ◽  
Leukemic Cells ◽  
Prolonged Survival ◽  
Hsp90 Inhibition ◽  
T315i Mutation

Abstract Development of mutations within the kinase domain is a major drug-resistance mechanism for tyrosine kinase inhibitors (TKIs) in cancer therapy. In CML (chronic myeloid leukemia), a disease driven by the constitutively active BCR-ABL oncoprotein, no available TKIs have been effective in treating patients with the BCR-ABL T315I mutation. Heat shock protein 90 (Hsp90) is a highly conserved, constitutively expressed molecular chaperone that facilitates folding of client proteins like BCR-ABL, and affects the stability of these proteins. Several labs have shown that Hsp90 inhibition in vitro results in the degradation of BCR-ABL T315I and induces potent killing of these cell lines. However, these results have not been demonstrated in animal models for BCR-ABL-induced CML and B-ALL (B-cell acute lymphoblastic leukemia, a disease that does not respond well to TKIs including imatinib and dasatinib). Thus, IPI-504, an orally administered Hsp90 inhibitor, was evaluated in murine models of CML and B-ALL. Treatment of mice with wild type (WT)- or T315I BCR-ABL-induced CML with IPI-504 resulted in BCR-ABL protein degradation and a decrease in circulating BCR-ABL positive cells. In response to treatment with vehicle the median survival time of WT and T315I CML mice is approximately 20 days. While the T315I CML mice were resistant to imatinib with a median survival of 21 days, IPI-504 (50 and 100 mg/kg, PO TIW) demonstrated dose-dependent prolonged survival of these mice by 30 and 70 days, respectively (p<0.001 for both doses). Both imatinib and IPI-504 similarly prolonged survival of mice with BCR-ABL-WT-induced CML. In the T315I CML mice prolonged survival of the IPI-504 treated cohort was associated with decreased peripheral blood BCR-ABL positive leukemia cells during treatment, less splenomegaly and improved pulmonary histopathlogy at necropsy. In CML mice receiving mixed BCR-ABL-WT- or T315I-transduced donor bone marrow cells, Hsp90 inhibition more potently suppressed T315I-expressing leukemia clones relative to the WT clones, consistent with in vitro studies where T315I BCR-ABL was more sensitive to IPI-504 induced degradation in cell lines than WT BCR-ABL. Combination treatment with IPI-504 and imatinib was more effective than either treatment alone in prolonging survival of mice bearing both WT and T315I leukemic cells. IPI-504 also significantly prolonged survival of B-ALL mice bearing the T315I mutation (p<0.001). These results provide a rationale for use of an Hsp90 inhibitor as a novel approach to overcoming resistance to TKIs as well as the potential for first line combination treatment in CML patients. The potential for IPI-504 to eliminate mutant kinases via Hsp90 inhibition provides a new therapeutic strategy for treating BCR-ABL-induced CML, ALL as well as other cancers resistant to treatment with TKIs.

FLT3 Immunotherapy Can Eliminate Engraftment of ALL Cells in NOD/SCID Mice.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 869-869
Author(s):  
Obdulio Piloto ◽  
Bao Nguyen ◽  
Patrick Brown ◽  
Kyu-Tae Kim ◽  
David Huso ◽  
...  
Keyword(s):  
Cell Lines ◽  
Scid Mice ◽  
Cytotoxic Agents ◽  
Radioactive Isotopes ◽  
Leukemic Cells ◽  
Pcr Analysis ◽  
Facs Analysis ◽  
Antibody Treatment

Abstract The class III receptor tyrosine kinase, FLT3, is expressed by over 90% of B-lineage acute lymphoblastic leukemias (ALL) blasts. In addition, it is expressed at extremely high levels in ALL patients with MLL-rearrangements or hyperdiploidy and sometimes mutated in these same patients. In this report, we investigated the effects of EB10, an anti-human FLT3 monoclonal antibody capable of preventing binding of FLT3 ligand (FL), on ALL cell lines and primary cells. In vitro studies, examining the ability of EB10 to inhibit FLT3 activation and downstream signaling in ALL cell lines and primary blasts, yielded variable results. In some cell lines FLT3 phosphorylation was inhibited and with it, downstream activation of pathways involving MAPK, AKT, and STAT5 phosphorylation. However, several cell lines actually exhibited FLT3 activation upon antibody treatment, possibly because of antibody-mediated receptor dimerization, and subsequent activation of downstream pathways. Nevertheless, through antibody-mediated cellular cytotoxicity (ADCC) such an antibody could still prove efficacious against leukemia cells in vivo. In fact, EB10 treatment significantly prolongs survival and/or reduces engraftment of several ALL cell lines and some primary ALL samples in NOD/SCID mice, even when EB10 treatment results in FLT3 activation of those cell lines in vitro. Moreover, FACS and PCR analysis of EB10 treated NOD/SCID mice surviving 150 days post leukemic cell injection revealed that FLT3 immunotherapy eliminated leukemic engraftment. The leukemic cells surviving EB10 treatment in the mice were characterized by FACS analysis and found to express lower levels of FLT3. To assess for resistance, cells surviving EB10 treatment were injected into NOD/SCID mice and treated with a single dose of EB10. FACS analysis revealed that these cells remain sensitive to EB10 treatment. Taken together, these data demonstrate that EB10 is cytotoxic to ALL blasts in vivo and EB10 treatment did not select for resistant clones. Such an antibody, either naked or conjugated to radioactive isotopes or cytotoxic agents, may prove useful in the therapy of infant ALL as well as childhood and adult ALL patients whose blasts typically express FLT3.

Improved Therapy of Multiple Myeloma by Combining Milatuzumab (Anti-CD74 Humanized mAb) and Bortezomib: In Vitro and In Vivo Results.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1176-1176
Author(s):  
Rhona Stein ◽  
David M. Goldenberg
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Leukemic Cells ◽  
Tumor Type ◽  
Term Survival ◽  
Significant Difference ◽  
Highly Effective ◽  
Ic50 Values

Abstract Background: The humanized anti-CD74 monoclonal antibody, milatuzumab (hLL1, or IMMU-115; Immunomedics, Inc, Morris Plains, NJ), is in clinical evaluation for therapy of multiple myeloma (MM) after preclinical evidence of activity in this tumor type (Stein et al, Blood2004;104:3705). Here we examine the ability of milatuzumab to increase the efficacy of drugs in MM cell lines. Methods: MTT cytotoxicity assays were performed on a panel of MM cell lines, including CAG, KMS11, KMS12-PE, and MC/CAR, to examine the effects of bortezomib, doxorubicin (dox), and dexamethasone (dex) alone and combined with milatuzumab or milatuzumab + crosslinking 2nd Ab (goat anti-human IgG, GAH). In vivo studies used a CAG-SCID mouse model of disseminated disease. Results: Without drugs, crosslinked milatuzumab, but not milatuzumab alone, yielded significant anti-proliferative effects on the four MM cell lines. In combination studies, crosslinked milatuzumab produced significant reductions in the IC50 values of the anti-MM drugs. For example, in CAG, milatuzumab+GAH decreased the IC50 values 58%, 78%, and 98% for bortezomib, dox, and dex, respectively (P=0.0034, 0.0073, and 0.078, respectively). In vivo, milatuzumab at 100 μg/injection, 2x weekly for 4 weeks, starting 1 day after injection of CAG cells, more than doubled the median survival time (MST) from 42 days in untreated CAG-bearing SCID mice to 103 days. Combination therapy with milatuzumab and bortezomib or dox was compared to milatuzumab alone, with treatments initiated 5 days after injection of CAG cells. Bortezomib alone (1.0 mg/kg) increased MST from 33 to 44 days (P=0.0021 vs. untreated). Treatment with milatuzumab alone (100 μg/mouse) increased the MST to 73 days (P<0.0001 vs. untreated). When bortezomib and milatuzumab treatments were combined, the MST increased to 93 days (P=0.0441 vs. milatuzumab and P=0.0065 vs. bortezomib). Thus, the combination of milatuzumab and bortezomib increased survival significantly compared to either single treatment. Given alone, dox yielded little or no effect on survival compared with untreated animals, and there was no significant difference between milatuzumab monotherapy and milatuzumab plus doxorubicin in this model. In contrast, a milatuzumabdox immunoconjugate was found to be a highly effective therapeutic agent, with all mice achieving long-term survival. The inhibition of the NF-κB survival pathway of B-leukemic cells by milatuzumab supports its complementary effects when combined with drugs having different mechanisms of action, such as bortezomib. Conclusions: The therapeutic efficacies of bortezomib, dox, and dex are enhanced in vitro in MM cell lines when given in combination with milatuzumab. In vivo, milatuzumab alone or especially in combination with bortezomib is highly effective in MM. (Supported in part by USPHS grant P01CA103985 from the NCI, and grants from the Thomas and Agnes Carvel Foundation and the Walter and Louise Sutcliffe Foundation.)

Role of Erythropoietin Receptor in t(12;21) Positive Acute Lymphoblastic Leukemia.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2792-2792
Author(s):  
Renate Panzer-Gruemayer ◽  
Gerd Krapf ◽  
Dominik Beck ◽  
Gerhard Fuka ◽  
Christian Bieglmayer ◽  
...  
Keyword(s):  
Cell Lines ◽  
Fusion Gene ◽  
Lymphoblastic Leukemia ◽  
Leukemic Cell ◽  
Erythropoietin Receptor ◽  
Molecular Signature ◽  
Leukemic Cells ◽  
Drug Induced ◽  
Induced Apoptosis

Abstract The chromosomal translocation t(12;21)(p13;q22) resulting in the TEL/AML1 (also known as ETV6/ RUNX1) fusion gene is the most frequent translocation in childhood B cell precursor (BCP) ALL. This type of ALL is characterized by a unique molecular signature, which includes the overexpression of the gene for the erythropoietin receptor (EpoR). So far, it is not known what causes the overexpression of the EpoR gene or whether it has any effect on the t(12;21) positive leukemia. We therefore aimed to evaluate potential mechanisms responsible for the upregulation of the EpoR in t(12;21) leukemias and to find out whether signalling via this receptor affects survival or proliferation of leukemic cells. In addition, we planned to explore signalling pathways linked to the respective effects and to elucidate relevant mechanisms that might be essential for cell survival. We first excluded the possibility that the EpoR expression is upregulated as a consequence of high Epo levels in the plasma that are induced by the patients’ low hemoglobin (Hb) levels. While Hb levels from patients with t(12;21)+ ALL were significantly lower compared to those with other subtypes of BCP ALL (median, 6,15g/dL and 7,9g/dL, respectively; p<0.001 Wilcoxon 2- sample test), which correlated with high Epo levels in the plasma, the extent of EpoR mRNA expression of leukemic cells was independent of the respective amount of Epo in the individual patient’s plasma. Next, the influence of Epo on t(12;21) + leukemic cell lines was evaluated and revealed a consistent time and dose dependent increase in proliferation (Epo concentrations 10, 50, 100U/ml for 72 hours) determined by 3H-Thymidine incorporation. This effect was abrogated upon addition of a blocking anti-EpoR antibody thereby confirming the specificity of EpoR signalling. Since Epo may have apoptosis-modulating potential in EpoR expressing malignant cells, we tested its influence on drug-induced apoptosis. For this purpose IC50 concentrations of drugs that are commonly used for the treatment of children with BCP ALL were used. A reduction of glucocorticoid (GC)-induced apoptosis by Epo was demonstrated in t(12;21)+ cell lines while no effect was seen in combination with other drugs or in t(12;21) negative cell lines. Preliminary data indicate that NF-kappa B as well as PI3K/Akt pathways are triggered by Epo, implying that they play a role in this rescue mechanism. Given that cell lines may have intrinsic changes, we are presently evaluating whether the observed results can also be reproduced in primary leukemic cells. In support of this assumption are results in a limited number of primary t(12;21)+ leukemias showing a superior survival (MTT assay) and reduced apoptosis rate to GC when cultured in the presence of Epo. These findings are in contrast to those in t(12;21) negative BCP ALLs. In conclusion, our data indicate that overexpression of EpoR in t(12;21) positive leukemias is not induced by low Hb, a feature that is generally observed in patients with this type of leukemia. Binding of Epo to its receptor in vitro leads to enhanced survival and negatively affects the sensitivity to GCs. Whether these findings have any implications on the treatment and care of patients with t(12;21)+ leukemia needs to be addressed in further studies. Financial support: OENB10720, FWF P17551-B14 and GENAU-CHILD Projekt GZ200.136/1 - VI/1/2005 to RPG.

Pharmacokinetic and Pharmacodynamic Effects of PEG-Asparaginase in Newly Diagnosed Childhood Acute Lymphoblastic Leukemia: Results from a Single Agent Window Study.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 860-860
Author(s):  
Inge M. Appel ◽  
Karin M. Kazemier ◽  
Anjo J.P. Veerman ◽  
Elisabeth van Wering ◽  
Monique L. Den Boer ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Clinical Response ◽  
Lymphoblastic Leukemia ◽  
Single Agent ◽  
Hazard Ratio ◽  
Leukemic Cells ◽  
Newly Diagnosed ◽  
Pharmacodynamic Effects

Abstract L-Asparaginase is an effective drug for treatment of children with acute lymphoblastic leukemia. The effectiveness is generally thought to result from a rapid depletion of asparagine in serum and cells. Several studies have shown that in vitro resistance to this drug is an independent prognostic factor in ALL. We investigated the clinical response of one in vivo dose of 1000 IU/m2 PEG-Asparaginase and its pharmacokinetic and pharmacodynamic effects in children with newly diagnosed ALL before the start of combination chemotherapy. 57 children (36M / 21F) were enrolled in the study: 2 pro B-ALL, 38 common/ pre B-ALL and 17 T-ALL. Genotyping of precursor B-ALL revealed 11 hyperdiploid, 8 TELAML1 positive, 2 BCRABL positive, no MLL rearrangement, 8 normal, 11 others. The clinical response to PEG-Asparaginase on day 0 (5 days after the PEG-Asparaginase infusion) was defined as good when the number of leukemic cells of peripheral blood was < 1 × 109/L, as intermediate when leukemic cells were 1-10 × 109/L, and as poor when leukemic cells were > 10 × 109/L. The in vivo window response was significantly related to immunophenotype and genotype: 26/38 common / pre B-ALL cases, especially those with hyperdiploidy and TELAML1 rearrangement, demonstrated a good clinical response compared to 8/17 T-ALL (p=0.01). Both BCRABL positive ALL cases showed a poor response (p=0.04). A poor in vivo clinical window response was related to in vitro resistance to L-Asparaginase (p=0.02) and both in vitro as well as in vivo response were prognostic factors for long-term event-free survival (Hazard ratio 6.4; p=0.004, and Hazard ratio 3.7; p=0.01, respectively). The L-Asparaginase activity in the serum was >100 IU/L for at least 15 days. The asparagine levels remained below the detection limit of 0.2 mM for at least 26 days with a concomitant rise in serum aspartate and glutamate. These findings confirm that PEG-Asparaginase will yield its pharmacodynamic effects for 2-4 weeks. After administration of one in vivo dose of 1000 IU/m2 PEG-Asparaginase no changes in apoptotic parameters or changes in intracellular levels of twenty amino acids in leukemic cells could be measured, in contradiction to the changes found after in vitro exposure. This may be explained by the rapid removal of apoptotic cells from the circulation in vivo. Otherwise it is possible that in vivo mesenchymal cells from the bone marrow supply leukemic blasts with asparagine in response to treatment with L-Asparaginase. Conclusion: The clinical response to one dose of 1000 IU/m2 PEG-Asparaginase intravenously is related to phenotype and genotype and predicts outcome. These results suggest that children with ALL with a poor clinical response to PEG-Asparaginase might benefit from a more intensive antileukemic therapy.

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