AT-101, a Pan-Inhibitor of Bcl-2 Family Anti-Apoptotic Proteins Antagonizes the Protective Effect Conferred by Nurselike Cells on Primary Chronic Lymphocytic Leukemia (CLL) Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2100-2100 ◽  
Author(s):  
Danelle F. James ◽  
Rebecca M. Mervis ◽  
Ruzbeh Mosadeghi ◽  
Thomas J. Kipps
Keyword(s):  
B Cells ◽  
Cell Viability ◽  
Single Agent ◽  
Leukemia Cells ◽  
Drug Induced ◽  
Accessory Cells ◽  
The Mean ◽  
Apoptotic Proteins

Abstract Despite the longevity of CLL B cells in vivo, in vitro CLL cells readily undergo apoptosis under conditions that support the growth of normal B cells. However, when leukemia cells are co-cultured with non-neoplastic accessory cells, such as those found in the marrow stroma and nurselike cells (NLC) that can differentiate from blood mononuclear cells they become resistant to spontaneous and drug induced cell death. These findings suggest that the defective apoptosis of the CLL B cells is not only ascribed to intrinsic defects in the neoplastic cell, but also to extrinsic factors that influence their behavior provided by the tumor microenvironment. As such, identification of molecular targets that link malignant B cells to supportive cells in the microenvironment may lead to new therapeutic avenues for CLL patients. For example, we have found that co-culture with NLC induces expression of Mcl-1, an anti-apoptotic protein of the Bcl-2 family in CLL cells. [Nishio M 2005] Induction of such prosurvival proteins by accessory cells may play a role disease aggressiveness and leukemia cell resistance to chemotherapy in vivo. AT-101 is a small molecule that mimics the inhibitory BH3 domain of endogenous antagonists of the Bcl-2 family anti-apoptotic proteins negating their cytoprotective role. Fluorescence polarization assays demonstrate that AT -101 binds to Bcl-2, BcL-XL, Bcl-W, and Mcl-1. AT-101 is cytotoxic to primary CLL cells in vitro and has been shown to have single agent activity in high risk CLL patients. Therefore, we hypothesized that inhibition of Mcl-1 by AT-101 may diminish the protection of NLC on CLL cells and may render the leukemia cells more sensitive to spontaneous and drug induced apoptosis. Primary CLL cells from 20 different patients were plated with or without NLCs, and cell viability was assessed overtime. Co-culture of CLL cells with NLC protected the CLL cells from undergoing spontaneous apoptosis. After 48 hours the average viability (+/− SEM) of CLL cells with NLC was 75% (+/− 4%) whereas the mean viability of CLL cultured alone was significantly lower at 59% (+/−5%) (p<0.02). In addition, we found that co-culture of CLL cells with NLC protected the leukemia cells from the cytotoxic effects of fludarabine (F-ara-A). For instance, after 48 hours after treatment with 10 μM F-ara-A the mean viability of the CLL cells was 28% (+/− 4%) whereas the viability of CLL cells cultured with NLC was 51% (+/−4%), a difference that was highly significant (p<0.001). In contrast, NLC could not protect leukemia cells from apoptosis induced by AT-101. For example, treatment of CLL cells with AT-101 at 5 μM, with or without NLC, resulted in CLL cell viability at 48 hours of only 40% (+/−7%.) or 30% (+/−7%.), respectively, a difference that was not statistically significant. AT-101 and F-ara-A were titrated in the setting of NLC and appeared to act additively in inducing apoptosis of CLL cells. In fact, NLC protection of CLL cells from fludarabine mediated cytotoxicity was negated in the presence of AT-101. Together our results describe a new therapeutic mechanism for targeting CLL interaction with the microenvironment. Specifically, a pan-inhibitor of Bcl-2 family anti-apoptotic proteins AT-101 antagonizes the protection of CLL by NLC to both spontaneous and fludarabine mediated apoptosis.

scholarly journals Bcl-2 antagonist apogossypol (NSC736630) displays single-agent activity in Bcl-2–transgenic mice and has superior efficacy with less toxicity compared with gossypol (NSC19048)

Blood ◽  
2008 ◽  
Vol 111 (6) ◽  
pp. 3211-3219 ◽  
Author(s):  
Shinichi Kitada ◽  
Christina L. Kress ◽  
Maryla Krajewska ◽  
Lee Jia ◽  
Maurizio Pellecchia ◽  
...  
Keyword(s):  
B Cells ◽  
Transgenic Mice ◽  
Parent Compound ◽  
Single Agent ◽  
Maximum Tolerated Dose ◽  
Low Grade ◽  
Altered Expression ◽  
Cell Counts

Abstract Altered expression of Bcl-2 family proteins plays central roles in apoptosis dysregulation in cancer and leukemia, promoting malignant cell expansion and contributing to chemoresistance. In this study, we compared the toxicity and efficacy in mice of natural product gossypol and its semisynthetic derivative apo-gossypol, compounds that bind and inhibit antiapoptotic Bcl-2 family proteins. Daily oral dosing studies showed that mice tolerate doses of apogossypol 2- to 4-times higher than gossypol. Hepatotoxicity and gastrointestinal toxicity represented the major adverse activities of gossypol, with apogossypol far less toxic. Efficacy was tested in transgenic mice in which Bcl-2 is overexpressed in B cells, resembling low-grade follicular lymphoma in humans. In vitro, Bcl-2–expressing B cells from transgenic mice were more sensitive to cytotoxicity induced by apogossypol than gossypol, with LD50 values of 3 to 5 μM and 7.5 to 10 μM, respectively. In vivo, using the maximum tolerated dose of gossypol for sequential daily dosing, apogossypol displayed superior activity to gossypol in terms of reducing splenomegaly and reducing B-cell counts in spleens of Bcl-2–transgenic mice. Taken together, these studies indicate that apogossypol is superior to parent compound gossypol with respect to toxicology and efficacy, suggesting that further development of this compound for cancer therapy is warranted.

Optimal NK Cell Expansion Depends on Accessory Cells, Synergy between Physiologic Concentrations of IL-2 and IL-15, and Umbilical Cord Blood (UCB) NK Cell Precursors Expand Better Than Adult NK Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3642-3642 ◽  
Author(s):  
Purvi Gada ◽  
Michelle Gleason ◽  
Valarie McCullar ◽  
Philip B. McGlave ◽  
Jeffrey S. Miller
Keyword(s):  
T Cells ◽  
B Cells ◽  
Nk Cells ◽  
Cell Expansion ◽  
Nk Cell ◽  
High Dose ◽  
Unexpected Result ◽  
Accessory Cells

Abstract Allogeneic NK cells may play a therapeutic role in treating patients with AML. We have previously shown that high dose cyclophosphamide (120 mg/kg × 1 day) and fludarabine (125 mg/m2 × 5 days) can clear lymphoid space and induce a surge of endogenous IL-15 to expand haploidentical NK cells obtained from CD3-depleted lymphapheresis products from adult donors. In this initial study, 5 of 19 patients achieved remissions and in vivo NK cell expansion. Limitations of this therapy includeinability of NK cells to expand in most patients,development of PTLD (in one patient) andinadequate disease control.We hypothesized that contaminating T cells could compete for NK cell expansion, that B-cells may contribute to PTLD, and that a 2-step NK cell purification method using CD3 depletion followed by CD56 selection (CliniMacs) may overcome these problems. We tested this in 9 patients with advanced AML. The purified NK cells, activated with 1000 U/ml IL-2 (16–20 hours), were infused 48 hours after the last fludarabine dose. Patients then received subcutaneous IL-2 (10 MU) every other day × 6 doses to expand NK cells in vivo. None of the 9 pts treated on this protocol achieved remission or exhibited evidence of in vivo expansion. Several studies were designed to investigate this unexpected result. First, we found that the more extensive processing resulted in approximately 1/3 the NK cell recovery compared to CD3 depletion alone (38±% viable NK cells vs. 91±2% respectively). In addition, we questioned whether the contaminating B cells and monocytes that were removed in the 2-step depletion strategy had served a critical role in NK cell activation or expansion. Cytotoxicity assays performed against K562 targets showed that the killing was about 3-fold higher with the purified (CD3-CD56+) product compared the CD3-depleted product alone (P=0.001 at E:T of 6.6:1). Proliferation, measured by a 6-day thymidine assay, was higher in proportion to the higher NK cell content. The only difference between the two NK products was their expansion after 14 days of culture, where the CD3-depleted product, with contaminating B-cells and monocytes, gave rise to greater NK cell expansion (14 ±3-fold) compared to the 2-step purified product (4.5±0.9, n=6, P=0.005). If this finding holds true in vivo, the co-infusion of accessory cells may be required for NK cell expansion. We next developed in vitro assays using very low concentrations (0.5 ng/ml) of IL-2 and IL-15 to understand their role in expansion. IL-2 or IL-15 alone induced low proliferation and the combination was synergistic. Lastly, UCB, a rich source of NK cell precursors, was compared to adult NK cells. In a short term proliferation assay, CD56+ NK cells stimulated with IL-2 + IL-15 expanded better from adult donors (61274±12999, n=6) than from UCB (20827± 6959, n=5, P=0.026) but there was no difference after 14 days in expansion culture suggesting that the only difference is in kinetics. However, UCB depleted of T-cells (enriching for NK cell precursors) exhibited higher fold expansion over 14 days under different culture conditions conducive to NK cell progenitors. In conclusion, NK cell expansion in vitro depends on cell source, IL-2 and IL-15 (increased in vivo after lymphoid depleting chemotherapy) as well as accessory cells. The role of these factors to enhance in vivo expansion is under clinical investigation to further exploit the NK cell alloreactivity against AML targets.

In Vitro and in Vivo Single-Agent Efficacy of Checkpoint Kinase 1 (Chk1) and 2 (Chk2) Inhibitor PF-0477736 (Pfizer) in B- and T-Acute Lymphoblastic Leukemia (ALL)

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1496-1496 ◽  
Author(s):  
Ilaria Iacobucci ◽  
Andrea Ghelli Luserna Di Rorà ◽  
Maria Vittoria Verga Falzacappa ◽  
Enrico Derenzini ◽  
Anna Ferrari ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Western Blot ◽  
Cell Lines ◽  
Single Agent ◽  
Time Dependent ◽  
Leukemia Cells ◽  
In Vivo Models

Abstract Abstract 1496 Introduction: Although progress in the treatment of ALL has been remarkable in children, in adults ALL still carries a dismal outcome. Thus, there is a need to improve therapeutic options. In the last years, selective inhibitors of Chk1 and/or Chk2 have been discovered, developed and entered in clinical trials. However, so far, they have not yet been investigated in leukemia. Chk1 and Chk2 are serine/threonine kinases that play a critical role in response to DNA damage both by halting the cell cycle through checkpoint activation and by actively repairing DNA. Here, we explored the in vitro and in vivo activity of single-agent inhibition of Chk1/2 by PF-0477736 in B- and T-progenitor ALL and we investigated potential biomarkers of functional inhibition. Methods: Human B (BCR-ABL1-positive: BV-173, SUPB-15; BCR-ABL1- negative: NALM-6, NALM-19, REH) and T (MOLT-4, RPMI-8402, CEM) leukemia cell lines were incubated with increasing concentrations of drug (5–2000 nM) for 24, 48 and 72 hours (hrs). Results: Inhibition of Chk1/2 resulted in a dose and time-dependent cytotoxicity with RPMI-8402 and BV-173 cells being the most sensitive (IC50 at 24 hrs: 57 nM and 82 nM, respectively), while NALM-6 cells the most resistant (IC50 at 24 hrs: 1426 nM)(WST-1 assay, Roche). Sensitivity did not correlate with p53 status (BV-173, SUPB-15, NALM-6 and NALM-19 cells were p53 wild-type whereas REH, MOLT-4, RPMI-8402 and CEM cells were p53 mutated) and with baseline levels of Chk1/2 and ATR/ATM phosphorylation, indicative of intrinsic genetic stress. Consistent with the viability results, Annexin V/Propidium Iodide (PI) staining analysis showed a significant increase of apoptosis at 24 and 48 hrs in a dose and time dependent manner coupled to increased proteolytic cleavage of PARP-1. In all sensitive cell lines in addition to the induction of apoptosis, Chk1/Chk2 inhibition induced DNA damage as demonstrated by the increased number of γH2AX foci (western blot and immunofluorescence analysis) and by a marked phosphorylation of Chk1 (ser317 and ser345). Moreover, PF-0477736 efficiently triggered the Chk1-Cdc25-Cdk1 pathway as soon as 24 hrs of treatment with a decrease of the inhibitory phosphorylation of Cdc25c (ser216) and Cdk1 (tyr15), leading to the abrogation of cell cycle arrest as confirmed by PI staining analysis at 6 and 24 hrs. The efficacy of PF-0477736 was thereafter demonstrated in primary leukemic blasts separated from 14 ALL patients. Based on the viability results at 24 hrs, 3 groups of patients were identified: very good responders, 5/14, 36% (IC50: 100–500 nM); good responders, 6/14, 43% (IC50: 600–1000 nM); poor responders, 3/14, 21% (IC50 > 1000 nM). By contrast, PF-0477736 did not show efficacy in primary cultures of normal bone marrow mononuclear cells, demonstrating its specificity for leukemia cells. We extended the in vitro and ex-vivo studies by assessing the efficacy of Chk inhibition in mice transplanted with T-lymphoid leukemia, demonstrating that PF-0477736 increases the survival of treated mice compared with mice treated with vehicle (p = 0.0016). Finally, in order to elucidate the mechanisms of action of PF-0477736 and to determine biomarkers of response, gene expression profiling analysis (Affymetrix GeneChip Human Gene 1.0 ST) was performed on treated leukemia cells and their untreated counterparts (DMSO 0.1%) after 24 hrs of incubation with concentrations equal to the IC50. Treatment resulted in a differential expression (p < 0.05) of genes involved in chromatin assembly, nucleosome organization and DNA packaging (e.g. Histone H1-H2A, 2B family clusters), DNA damage (DDIT3, GADD34 and GADD45a) and apoptosis (e.g. CDKN1A, BAX, FAS, BTG1), confirming that PF-0477736 contributes to checkpoint replication abrogation, accumulation of DNA damage and subsequent apoptosis in leukemia cells. Interestingly, N-Myc and c-Myc expression strongly decreased after treatment, as also confirmed by western blot analysis, suggesting that a negative feedback loop may exist between Chk induction and Myc expression. Conclusions: Together, these results demonstrate the efficacy of PF-0477736 both in vitro and in vivo models of ALL, arguing in favor of its future clinical evaluation in leukemia. Supported by ELN, AIL, AIRC, Fondazione Del Monte di Bologna-Ravenna, PRIN2009, PIO program, Programma Ricerca Regione-Università 2007–2009. PF-0477736 provided by Pfizer. Disclosures: Baccarani: ARIAD, Novartis, Bristol Myers-Squibb, and Pfizer: Consultancy, Honoraria, Speakers Bureau. Martinelli:NOVARTIS: Consultancy, Honoraria, Speakers Bureau; BMS: Consultancy, Honoraria, Speakers Bureau; PFIZER: Consultancy; ARIAD: Consultancy.

scholarly journals Cellular and genetic control of antibody responses. V. Helper T-cell recognition of H-2 determinants on accessory cells but not B cells.

1979 ◽  
Vol 149 (5) ◽  
pp. 1208-1226 ◽  
Author(s):  
A Singer ◽  
K S Hathcock ◽  
R J Hodes
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Helper T Cells ◽  
Cell Recognition ◽  
Helper T Cell ◽  
T Cell Recognition ◽  
Accessory Cells

Requirements for helper T-cell recognition of H-2 determinants expressed on adherent accessory cells and on B cells was individually assessed in the anti-hapten PFC responses to TNP-KLH. Complicating allogeneic effects were minimized or avoided by the use of helper T cells from normal F1 hybrids, parent leads to F1 chimeras, and F1 leads to parent chimeras. The results of both in vitro and in vivo experiments demonstrated that: (a) helper T cells are not required to recognize the identical H-2 determinants on both accessory cells and B cells; (b) helper T cells are required to recognize K or I-A region-encoded determinants expressed on accessory cells; (c) no requirement was observed in vitro or in vivo for helper T-cell recognition of B-cell-expressed H-2 determinants; and (d) no requirement was observed for H-2 homology between accessory cells and B cells. The absence of required helper T-cell recognition of the identical H-2 determinants on both accessory cells and B cells was demonstrated in two ways: (a) naive of KLH-primed (A x B)F1 hybrid helper T cells collaborated equally well with B cells from either parentA or parentB in the presence of accessory cells from either parent; (b) A leads to (A x B)F1 chimeric spleen cells depleted of accessory cells collaborated equally well with accessory cells from either parentA or parentB, even though the B cells only expressed the H-2 determinants of parentA. A requirement for helper T-cell recognition of K or I-A region-encoded H-2 determinants on accessory cells was also demonstrated in two ways: (a) (A x B)F1 leads to parentA chimeric spleen cells depleted of accessory cells collaborated with accessory cells from parentA but not parentB; and (b) (A x B)F1 leads to parentA chimeric helper T cells collaborated with normal F1 B cells only in the presence of parental or recombinant accessory cells that expressed the K or I-A region-encoded determinants of parentA. Although restricted in their ability to recognize H-2 determinants on accessory cells, it was demonstrated both in vitro and in vivo that (A x B)F1 leads to parentA chimeric helper T cells were able to collaborate with B cells from either parentA or parentB. In vitro in the presence of accessory cells from parentA, (A x B)F1 leads to parentA chimeric helper T cells collaborated equally well with B cells from either parent. In addition, the inability of (A x B)F1 leads to parentA chimeric helper T cells to collaborate with (B + accessory) cells from parentB was successfully reversed by the addition of parentA SAC as added accessory cells. In vivo, upon the addition of parentA accessory cells, (A x B)F1 leads to parentA chimeric helper T cells collaborated with parentB B cells in short-term adoptive transfer experiments.

scholarly journals Role of accessory cells in B cell activation. III. Cellular analysis of primary immune response deficits in CBA/N mice: presence of an accessory cell-B cell interaction defect.

1980 ◽  
Vol 152 (5) ◽  
pp. 1194-1309 ◽  
Author(s):  
H S Boswell ◽  
M I Nerenberg ◽  
I Scher ◽  
A Singer
Keyword(s):  
T Cells ◽  
B Cells ◽  
B Cell ◽  
Genetic Defect ◽  
Accessory Cell ◽  
Male Mice ◽  
Accessory Cells ◽  
Antigen Presenting

The effect of the X-linked CBA/N genetic defect on the ability of mice to generate primary responses to thymic-dependent and thymic-independent antigens was assessed by comparing the ability of abnormal (CBA/N x DBA/2)F1 male mice and normal (DBA/2 x CBA/N)F1 male mice to generate 2,4,6-trinitrophenyl (TNP)-specific plaque-forming cell responses to TNP-keyhole limpet hemocyanin (KLH), TNP-conjugated Ficoll (TNP-Ficoll), TNP-Brucella abortus (BA), and TNP-lipopolysaccharide (LPS). The reciprocal F1 combinations used in this study differ genetically only in the origin of their X chromosome, but differ immunologically in that (CBA/N x DBA/2)F1 male mice express all the CBA/N immune abnormalities, whereas (DBA/2 x CBA/N)F1 male mice are immunologically normal. Analysis of thymic-dependent responses to TNP-KLH revealed that abnormal F1 mice were capable of generating primary responses in vivo to high doses of TNP-KLH, but failed to generate responses to suboptimal doses of TNP-KLH that were still immunogenic for normal F1 mice. Furthermore, under limiting in vitro micro-culture conditions, the abnormal F1 mice failed to generate primary thymic-dependent responses to any dose of TNP-KLH, even though under the identical conditions normal F1 mice consistently responded to a wide antigen dose range. The cellular basis of the failure of abnormal F1 mice to respond in vitro to TNP-KLH was investigated by assaying the ability of purified populations of accessory cells, T cells, and B cells from these mice to function in responses to TNP-KLH. The results of these experiments demonstrated that helper T cells and antigen-presenting accessory cells from abnormal F1 mice were competent and functioned as well as the equivalent cell populations from normal F1 mice. Instead, the failure of CBA/N mice to generate primary in vitro responses to TNP-KLH was solely the result of a defect in their B cell population such that B cells from these mice failed to be triggered by competent helper T cells and/or competent accessory cells. Similarly, the failure of abnormal F1 mice to respond either in vivo or in vitro to TNP-Ficoll was not the result of defective accessory cell presentation of TNP-Ficoll, but was the result of the failure of B cells from these mice to be activated by competent TNP-Ficoll-presenting accessory cells. In contrast to the failure of B cells from abnormal F1 mice to be activated in vitro in response to either TNP-KLH or TNP-Ficoll, B cells from abnormal F1 mice were triggered to respond to TNP-BA and TNP-LPS, antigens that did not require accessory cell presentation. The specific failure of B cells fron abnormal F1 mice to be activated in responses that required antigen-presentation by accessory cells suggested the possibility that the X-linked CBA/N genetic defect resulted in B cell populations that might be deficient in their ability to interact with antigen-presenting accessory cells...

scholarly journals Novel Bruton's Tyrosine Kinase Inhibitor Bgb-3111 Demonstrates Potent Activity in Mantle Cell Lymphoma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5374-5374 ◽  
Author(s):  
Carrie J Li ◽  
Yang Liu ◽  
Taylor Bell ◽  
Jack Wang ◽  
Hui Guo ◽  
...  
Keyword(s):  
Tumor Growth ◽  
Cell Viability ◽  
Research Funding ◽  
Cell Lymphoma ◽  
Single Agent ◽  
Mantle Cell ◽  
Btk Inhibitor ◽  
Dose Dependent

Abstract Background: Aberrant B-cell receptor signaling is an important contributor to lymphomagenesis in mantle cell lymphoma (MCL). Bruton's Tyrosine Kinase (BTK), a component of the BCR signaling axis, has been validated as a clinically relevant target, and BTK inhibitor ibrutinib received FDA approval for treatment of MCL in 2013. Growing concerns that single agent ibrutinib exerts off-target effects that interfere with other treatments such as rituximab-induced antibody-dependent cell cytotoxicity limit its utility in combination treatments. In this study, we assessed the in vitro and in vivo effects of BGB-3111in MCL models. Methods: We performed cell viability assays with BGB-3111 treated MCL cell lines to determine inhibition of cellular proliferation. The same assays were conducted on primary human MCL cells and patient-derived xenograft (PDX) tumor samples. Dose-dependent inhibition of BTK auto-phosphorylation and inhibition of downstream targets such as PLC-γ were determined by phospho-protein immunoblotting and immunoprecipitation. A reverse-phase protein assay (RPPA) was conducted on BGB-3111-treated Mino cells to evaluate changes in MCL oncogenic signaling. Induction of apoptosis in MCL cells treated with increasing doses of BGB-3111 was quantified using flow cytometry. For in vivo experiments, an ibrutinib-sensitive MCL PDX mouse model was treated with 50 mg/kg/day BGB-3111 and monitored for mean tumor burden and survival. Results: BGB-3111 potently inhibited cell viability in a panel of MCL cell lines, with an activity range of 1-10 uM, and induced apoptosis in a dose-dependent manner in several MCL cell lines.BGB-3111 treatment of MCL cells demonstrated a dose-dependent decrease in p-BTK (Y223) and inhibition of downstream effectors without impacting total protein levels, while RPPA revealed upregulation of the PI3K-Akt signaling axes. In addition, BGB-3111 treatment did not impact phosphorylation of off-target kinases affected by ibrutinib treatment. In vivo, BGB-3111 suppressed tumor growth and prolonged tumor survival in BGB-3111 treated mice. Conclusion: The second generation BTK inhibitor BGB-3111 demonstrates selectivity for BTK in vitro and BTK inhibition in vivo. BGB-3111-treated PDX mouse models examining survival, tumor growth, and other factors point to BGB-3111 as an effective single agent BGB-3111 is being investigated in Phase I clinical trials. Disclosures Wang: Beigene: Employment. Wang:Asana BioSciences: Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Dava Oncology: Honoraria; Acerta: Consultancy, Research Funding; Kite Pharma: Research Funding; BeiGene: Research Funding; Asana biosciences, Beigene, Celgene, Juno, Kite, Onyx, Pharmacyclics: Research Funding; Juno Therapeutics: Research Funding.

Diallyl sulfide inhibits murine WEHI-3 leukemia cells in BALB/c mice in vitro and in vivo

2009 ◽  
Vol 28 (12) ◽  
pp. 785-790 ◽  
Author(s):  
Fu-Shun Yu ◽  
Chih-Chung Wu ◽  
Chi-Tsai Chen ◽  
Shang-Pang Huang ◽  
Jai-Sing Yang ◽  
...  
Keyword(s):  
B Cells ◽  
Allium Sativum ◽  
Leukemia Cells ◽  
T And B Cells ◽  
Control Groups ◽  
Diallyl Sulfide ◽  
In Vivo Effects ◽  
Dose Dependent

It is well documented that enhanced garlic (Allium sativum) consumption leads to decrease in the cancer incidences. Diallyl sulfide (DAS), one of the components of garlic, induces cytotoxicity and apoptosis in many cancer cell lines. The present studies are focused on the in vivo effects of DAS on leukemia WEHI-3 cells in the BALB/c mice. We examined the effects of DAS on the cytotoxicity of WEHI-3 cells and results indicated that DAS decreased the percentage of viable WEHI-3 cells and these effects are dose-dependent. We examined the effects of DAS on WEHI-3 in vivo and the results indicated that DAS decreased the percentage of Mac-3 and CD11b, indicating that the differentiation of the precursor of macrophage cells was inhibited. DAS stimulated the percentage of CD3 and CD19, indicating that the differentiation of the precursor of T and B cells promoted. The weights of liver and spleen indicated that DAS decreased the weight of these organs after being compared to the control groups. One of the major characteristic of WEHI-3 leukemia is the enlarged spleen in murine after intraperitoneal (i.p.) injection of WEHI-3 cells. In conclusion, DAS affects WEHI-3 cells both in vitro and in vivo.

R-(−)-Gossypol (AT101) Binds to Bcl-2 Family Proteins and Induces Apoptosis in CLL.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2978-2978
Author(s):  
Carlos E. Prada ◽  
Januario E. Castro ◽  
Dayong Zhai ◽  
Shinichi Kitada ◽  
John C. Reed ◽  
...  
Keyword(s):  
Cell Death ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Programmed Cell Death ◽  
Binding Affinity ◽  
Lymphocytic Leukemia ◽  
Leukemia Cells ◽  
Mutational Status ◽  
Apoptotic Proteins

Abstract Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of monoclonal B cells in the blood, secondary lymphoid tissues, and marrow. The leukemia cells primarily are arrested in the G0/G1 phase of the cell cycle and appear resistant to programmed cell death. Several anti-apoptotic proteins are over expressed in CLL and this correlates with resistance to treatment, disease progression and overall poor prognosis. Proteins in the Bcl-2 family are central regulators of programmed cell death, and members that inhibit apoptosis, such as Bcl-X(L), Bcl-2, and Mcl-1, are overexpressed in many cancers including CLL and contribute to tumour initiation, progression and resistance to therapy. Mcl-1 is of particular interest because this molecule appears to be regulated by Nurse-like Cells and other stromal cells that promote survival of CLL cells in vitro and very likely also in vivo. These proteins enhance the resistance of CLL cells to spontaneous and/or drug-induced apoptosis primarily by interacting with, and antagonizing the activity of mitochondria membrane pro-apoptotic proteins such as Bax and Bak. The protein-protein interaction of Bcl-2 family members is critical for their activity, and these interactions are governed by binding to the BH3 domain. Racemic gossypol is found in cotton seeds and has been studied as a cytotoxic agent in cancer cell lines and in clinical studies in patients with a large variety of cancers. The antitumor activity of racemic gossypol appears to reside principally in the R-(−)-enantiomer (AT101), with reduced activity observed for the S-(+)-enantiomer. AT101 is an antagonist of the BH3-binding groove of the Bcl-2 family of proteins that can inhibit the interactions of these proteins with pro-apoptotic molecules. We examined whether AT101 could induce apoptosis in Chronic Lymphocytic Leukemia (CLL) and its ability to bind in vitro anti-apoptotic molecules from the Bcl-2 family. Using a Fluorescence Polarization Assay (FPA) we studied the competitive binding affinity of AT101 to Bcl-2 family member proteins. We observed that both, racemic gossypol and AT101 had comparable affinity for Bck-2, Bcl-B, Bfl-1 with EC50=0.6 to 10 μM range. AT101 had a stronger binding affinity to Bcl-X(L) (EC50=0.998 μM vs. 3.084 μM for racemic gossypol), and to Mcl-1 (EC50= 0.52μM vs. 1.07μM for racemic gossypol). CLL cells were incubated with AT101 for 48 hrs at different concentrations. We observed that leukemia cells were induced to undergo apoptosis in a time and dose dependent manner and that this effect was independent of ZAP-70 expression or IgVH gene mutational status (IC50= 2μM). Cells undergoing apoptosis showed PARP-1 cleavage and upregulation of pro-apoptotic molecules such as Bid, p53, as well as downregulation of Mcl-1. These results indicate that AT101 has stronger pan-specific binding affinity for Bcl-2 family proteins than racemic gossypol, in particular to Mcl-1 and Bcl-X(L), and that this compound induces apoptosis in CLL B cells independently of ZAP-70 expression or IgVH gene mutational status. Because of these encouraging results a clinical trial using AT101 in CLL patients with high-risk features is currently open at our institution.

The Humanized Anti-CD40 Antibody SGN-40 Inhibits Tumor Growth in LAGκ-1A, a CD40+ Mouse Model of Human Multiple Myeloma.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3506-3506 ◽  
Author(s):  
Richard A. Campbell ◽  
Melinda S. Gordon ◽  
Eric Sanchez ◽  
Haiming Chen ◽  
Lauren Turker ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
B Cells ◽  
Tumor Growth ◽  
Murine Model ◽  
Single Agent ◽  
Mouse Serum ◽  
Igg Antibody ◽  
Inhibition Of Tumor Growth

Abstract CD40 is a TNF receptor found on the cell surface of mature B cells (B lymphocytes) and most B-cell malignancies including multiple myeloma (MM). SGN-40 is a high-affinity, humanized monoclonal antibody that targets the CD40 antigen. Recently, it has been shown that SGN-40 decreases the proliferation of malignant B cells by partial agonistic signaling and effector functions in vitro. In this study, we examined the anti-MM effects of SGN-40 in vivo using a CD40+ SCID-hu murine model of human myeloma, LAGκ-1A. Each immunodeficient (SCID) mouse was implanted with a 2.0 – 4.0 mm3 LAGκ-1A tumor fragment into the left hind limb muscle. The tumor was allowed to grow for 14 days at which time human IgG levels were detectable in the mouse serum. Mice were then randomly assigned to one of four SGN-40 treatment groups (6 mice per treatment group). SGN-40 was administered via intraperitoneal injection twice per week at doses of 0.1, 0.3, 1, and 3 mg/kg. Control mice were given a control IgG antibody (3 mg/kg) using the same schedule. Mice receiving the higher doses of SGN-40 showed marked inhibition of tumor growth (0.3 mg/kg, P < 0.02; 1 mg/kg, P < 0.03; and 3 mg/kg, P < 0.04) and reduction of paraprotein levels (1 mg/kg, P < 0.05; and 3 mg/kg, P < 0.03) compared to mice receiving control antibody. At the lowest dose of SGN-40 evaluated (0.1 mg/kg) a slight inhibition of tumor growth was observable, but there was no effect on human paraprotein. Treatment with SGN-40 was not associated with any observed toxicity. Based on these data with SGN-40 monotherapy, we are currently investigating the antitumor activity of SGN-40 plus bortezomib as well as other available anti-MM agents using our in vivo SCID-hu myeloma murine model. These data for single-agent SGN-40 are encouraging and support testing SGN-40 both alone and in combination regimens to treat MM patients.

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