Dipeptidyl Peptidase 2 - A Pro-Survival Molecule in Chronic Lymphocytic Leukemia.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4964-4964
Author(s):  
Alexey V. Danilov ◽  
Olga V. Danilova ◽  
Andreas K. Klein ◽  
Brigitte T. Huber
Keyword(s):  
Cell Cycle ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Serine Protease ◽  
Specific Inhibitor ◽  
Dipeptidyl Peptidase ◽  
Chemotherapeutic Agents ◽  
Lymphocytic Leukemia ◽  
Quiescent State ◽  
Induced Apoptosis

Abstract Chronic lymphocytic leukemia (CLL) is a unique malignancy characterized by persistent accumulation of quiescent B-cells. Disruption of survival pathways leads to apoptosis and renders CLL cells sensitive to chemotherapeutic agents. As clinical outcomes in CLL are heterogeneous, it is important to better predict prognosis in each individual case of the disease. In this respect, detection of somatic mutations in the immunoglobulin variable heavy chain (IgVH) genes has become the gold standard. Overexpression of ZAP-70 (Zeta-chain associated protein kinase - 70 kDa) by CLL cells correlates with unmutated IgVH genes and predicts poor outcome in B-CLL. Dipeptidyl Peptidase 2 (DPP2) is a newly discovered serine protease which maintains lymphocytes in a quiescent state (G0). In this study we investigated whether DPP2 is involved in cell cycle control in B-CLL. 38 patients with B-CLL and 20 healthy controls were included in the study. Median age of CLL patients was 67 years. Median time from diagnosis to enrollment in the study was 102 months. 21 patients (55.3%) received treatment before enrollment in the study. Standard Ficoll-Hypaque techniques were used to isolate peripheral blood mononuclear cells (PBMC) from healthy donors and CLL patients. CLL cells were isolated to >98% purity by means of a MoFlo using CD19−specific antibodies. Cells were treated with Val-boro-Pro, a pan-inhibitor of DPP, or with DPP2-specific inhibitor (DSI), incubated for 16 hours and stained with propidium iodide and Annexin V. Expression of CD38 was assessed by flow cytometry, DPP2 and ZAP-70 - by real-time reverse-transcription polymerase chain reaction, Bcl-2 and p27 - by western blot analysis. We report that CLL B-cells expressed higher levels of DPP2 mRNA than normal B-cells. Inhibition of DPP2 in PBMC with VbP or DSI resulted in caspase-dependent apoptosis. In individuals with CLL, death of B-lymphocytes (and rescue by caspase inhibitors) was observed in 22 cases (57.9%). In the remaining 16 cases (42.1%) malignant B-cells did not undergo apoptosis upon inhibition of DPP2 with either VbP or DSI. We found that CLL cells resistant to DPP2 inhibition-induced apoptosis expressed higher levels of ZAP-70. Meanwhile, protein levels of p27 (cell cycle inhibitor) were decreased in resistant CLL. These patients exhibited worse disease prognosis such as shorter treatment-free period (p<0.001), frequent episodes of treatment failure and faster disease progression. Between the two groups, we identified no differences in expression of Bcl-2. CLL B-cells express higher levels of DPP2, a serine protease involved in maintenance of G0 which may serve as a survival factor in CLL B-cells. Resistance vs. susceptibility to DPP2 inhibition-induced apoptosis can be employed as a prognostic factor in CLL.

Theophylline synergizes with chlorambucil in inducing apoptosis of B- chronic lymphocytic leukemia cells

Blood ◽  
1996 ◽  
Vol 88 (6) ◽  
pp. 2172-2182 ◽  
Author(s):  
F Mentz ◽  
MD Mossalayi ◽  
F Ouaaz ◽  
S Baudet ◽  
F Issaly ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Apoptotic Cell ◽  
Protein A ◽  
Phosphodiesterase Inhibitor ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Lymphocytic Leukemia ◽  
Interleukin 4 ◽  
Induced Apoptosis

We tested the effects of theophylline, a phosphodiesterase inhibitor inducing intracellular accumulation of cyclic adenosine monophosphate (cAMP), on malignant B cells from 15 patients with B-chronic lymphocytic leukemia (B-CLL). We observed a large increase in apoptotic cell numbers (mean, 90% v 20% in medium alone) in the presence of theophylline (100 micrograms/mL) or chlorambucil (10 mumol/L) after 72 hours of incubation. Maximal apoptosis (90%) was reached after 36 hours when the two drugs were used together at fourfold lower concentrations, indicating a synergistic effect; no effect was observed with normal B cells, suggesting that the combination might have therapeutic interest. Chlorambucil induced intracellular Ca+2 influx, pointing to the involvement of two signaling pathways that might explain its synergy with theophylline through their effects on oncogenes. The expression of bcl-2 protein, a proto-oncogene inhibiting apoptosis, decreased after incubation with the drugs, while c-myc, recently described as having a potent role in apoptosis, was overexpressed. For p53 we observed an overexpression in the presence of chlorambucil or both theophylline- chlorambucil and a decrease after theophylline incubation. Chlorambucil- and theophylline-induced apoptosis was partially inhibited by interleukin-4 (IL-4), which also abrogated the effects on oncogene expression. These results provide insight into the mechanisms underlying B-CLL apoptosis and suggest that the theophylline- chlorambucil combination may be of therapeutic value in this setting.

Macrophage Migration Inhibitory Factor (MIF) Promotes the Development of Murine Chronic Lymphocytic Leukemia (CLL)

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 27-27
Author(s):  
Nina Reinart ◽  
Malgorzata Ciesla ◽  
Cornelia Rudolph ◽  
Astrid Stein ◽  
Guenter Krause ◽  
...  
Keyword(s):  
Cell Cycle ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Migration Inhibitory Factor ◽  
Macrophage Migration Inhibitory Factor ◽  
Lymphocytic Leukemia ◽  
Inhibitory Factor ◽  
Macrophage Migration ◽  
Spontaneous Apoptosis ◽  
Apoptosis Pathway

Abstract Introduction: Tumor formation results from a complex interplay between genetic/epigenetic alterations, cell cycle dysregulation and promotion by the tumor environment. Stimulation by extracellular survival factors is important for chronic lymphocytic leukemia (CLL), since the leukemic cells undergo spontaneous apoptosis when removed from their normal milieu. Since preliminary experiments demonstrated that macrophage migration inhibitory factor (MIF), a chemokine-like proinflammatory mediator and an intracellular regulator of growth and apoptosis, is overexpressed in human CLL, we investigated whether MIF participates in the pathogenesis of murine CLL. Methods: We studied the role of MIF in CLL by crossing the Eμ-TCL1-transgenic mouse model with MIF knockout (MIF−/−) mice. B-cell-specific overexpression of T cell leukemia-1 (TCL1) leads to accumulation and proliferation of IgM+/CD5+ mature B-cells via activation of AKT. This results in a CLL-like disease with peripheral lymphocytic leukemia, lymphadenopathy, splenomegaly, BM infiltration and premature death after 8–15 months. TCL1+/wtMIF−/− and TCL1+/wtMIF+/+ mice were compared with respect to leukemia development, tumor burden, cytogenetics and survival. Results: The MIF receptors CD74/CD44 and CXCR2 are expressed on murine B-cells. TCL1+/wtMIF+/+ mice exhibited increased numbers of IgM+/CD5+ B-cells already in the preleukemic phase at month 3 and developed overt leukemia (WBC > 20G/l) 3 months earlier than their MIF−/− counterparts (p = 0.02). Leukemia load at 12 months of age as measured by hepatosplenomegaly was increased in TCL1+/wtMIF+/+ animals and lymphatic organs were densely infiltrated by small, mature lymphocytes. The accelerated disease progression in the presence of MIF translated into a median survival which was 60 days shorter than in the absence of MIF (TCL1+/wtMIF+/+ 400 days, TCL1+/wtMIF−/− 460 days, p = 0.04). SKY analysis in leukemic splenocytes yielded various complex genetic aberrations with trisomies (e.g. +15), tetraploidy, translocations and deletions. Overexpression of tp53 due to the presence of an inactivating mutation in the p53 gene was found more frequently in TCL1+/wtMIF+/+ than in TCL1+/wtMIF−/− animals. Although the rates of DNA-damage-induced apoptosis in pre-leukemic and leukemic mice ex vivo were not significantly different between the genotypes, this defect in the p53-dependent apoptosis pathway corresponded with a reduced rate of spontaneous apoptosis in spleens of leukemic TCL1+/wtMIF+/+ animals. Conclusions: Our experience with the Eμ-TCL-1-transgenic mice shows that this model is suitable for the identification of novel regulators of CLL-like disease. We provide genetic proof that MIF acts to promote the early preleukemic and the leukemic phase of TCL1-induced CLL and thereby identify MIF as a novel regulator of CLL pathogenesis. Ongoing efforts are focussing on further characterizing the differences in pathology, the activation of the AKT pathway and cell cycle control between TCL1+/wtMIF−/− and TCL1+/wtMIF+/+ mice.

scholarly journals A Detailed Analysis of Parameters Supporting the Engraftment and Growth of Chronic Lymphocytic Leukemia Cells in Immune-Deficient Mice

2021 ◽  
Vol 12 ◽  
Author(s):  
Piers E. M. Patten ◽  
Gerardo Ferrer ◽  
Shih-Shih Chen ◽  
Jonathan E. Kolitz ◽  
Kanti R. Rai ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Intraperitoneal Administration ◽  
Human Condition ◽  
Lymphocytic Leukemia ◽  
Lymphoid Tissues ◽  
Quiescent State

Patient-derived xenograft models of chronic lymphocytic leukemia (CLL) can be created using highly immunodeficient animals, allowing analysis of primary tumor cells in an in vivo setting. However, unlike many other tumors, CLL B lymphocytes do not reproducibly grow in xenografts without manipulation, proliferating only when there is concomitant expansion of T cells. Here we show that in vitro pre-activation of CLL-derived T lymphocytes allows for a reliable and robust system for primary CLL cell growth within a fully autologous system that uses small numbers of cells and does not require pre-conditioning. In this system, growth of normal T and leukemic B cells follows four distinct temporal phases, each with characteristic blood and tissue findings. Phase 1 constitutes a period during which resting CLL B cells predominate, with cells aggregating at perivascular areas most often in the spleen. In Phase 2, T cells expand and provide T-cell help to promote B-cell division and expansion. Growth of CLL B and T cells persists in Phase 3, although some leukemic B cells undergo differentiation to more mature B-lineage cells (plasmablasts and plasma cells). By Phase 4, CLL B cells are for the most part lost with only T cells remaining. The required B-T cell interactions are not dependent on other human hematopoietic cells nor on murine macrophages or follicular dendritic cells, which appear to be relatively excluded from the perivascular lymphoid aggregates. Notably, the growth kinetics and degree of anatomic localization of CLL B and T cells is significantly influenced by intravenous versus intraperitoneal administration. Importantly, B cells delivered intraperitoneally either remain within the peritoneal cavity in a quiescent state, despite the presence of dividing T cells, or migrate to lymphoid tissues where they actively divide; this dichotomy mimics the human condition in that cells in primary lymphoid tissues and the blood are predominately resting, whereas those in secondary lymphoid tissues proliferate. Finally, the utility of this approach is illustrated by documenting the effects of a bispecific antibody reactive with B and T cells. Collectively, this model represents a powerful tool to evaluate CLL biology and novel therapeutics in vivo.

Motexafin Gadolinium Induces Apoptosis in Lymphoid Cell Lines and Demonstrates Enhanced Biological Activity with Akt Kinase Inhibitors.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3406-3406
Author(s):  
Louie Naumovski ◽  
Jason Ramos ◽  
Jun Chen ◽  
Mint Sirisawad ◽  
David Lucas ◽  
...  
Keyword(s):  
Cell Death ◽  
Chronic Lymphocytic Leukemia ◽  
Cell Lines ◽  
Specific Inhibitor ◽  
Annexin V ◽  
Lymphocytic Leukemia ◽  
Jurkat Cells ◽  
Motexafin Gadolinium ◽  
Akt Phosphorylation ◽  
Induced Apoptosis

Abstract Motexafin gadolinium (MGd, Xcytrin®) is a tumor-selective redox mediator that catalytically oxidizes intracellular reducing metabolites and produces reactive oxygen species (ROS). In this report, we demonstrate that MGd induces apoptosis or growth inhibition in several hematopoietic tumor-derived cell lines and tumor cells from patients with chronic lymphocytic leukemia. Lymphoma (HF-1, Ramos, DHL-4, DB, Hut78 and Raji) and leukemia (Jurkat, HL-60) cell lines were cultured in RPMI 1640 media with 10% heat inactivated fetal bovine serum with or without 50 uM MGd. MGd inhibited the growth of 6 of the cell lines (HF-1, Ramos, HL-60, DHL-4, Jurkat and DB) and was cytotoxic to HF-1. ROS were implicated in MGd-induced cell death since their presence was detected by dichlorofluorescein diacetate staining and peroxiredoxin oxidation in MGd treated HF-1 cells that undergo apoptosis, but not in Jurkat cells that do not undergo MGd-induced apoptosis. MGd triggered an apoptotic pathway in HF-1 cells as demonstrated by loss of mitochondrial membrane potential, release of cytochrome c from mitochondria, activation of caspases, cleavage of PARP and annexin-V binding. MGd also induced cell death and activated caspases in vitro in primary chronic lymphocytic leukemia cells. Protein lysates from cultured cell lines (HF-1, Ramos) were subjected to immunoblot analysis to determine caspase cleavage patterns, and the phosphorylation status of Akt, a kinase that regulates survival pathways. In MGd treated HF-1, phospho-Akt protein levels initially increased 2–3 fold between 30 min and 1 hr (n=4) and then decreased to 40–50% of control levels by 24–48 hrs (n=4). The drop in phospho-Akt protein coincided with an increase in apoptotic cell death as indicated by morphology, staining with Annexin-V and activation of caspases. Addition of a specific inhibitor of Akt phosphorylation (SH-5) reduced Akt phosphorylation in MGd treated HF-1 cells by 90% and enhanced the cytotoxic effect of MGd. In Ramos cells, which do not undergo apoptosis when treated with MGd, co-treatment with MGd and SH-5 decreased phospho Akt levels by only 15% and did not result in cytotoxicity. These data point to a potential role for Akt in MGd-induced apoptosis and suggest that MGd activity may be enhanced by inhibition of Akt. These data show that the pro-apoptotic effects of MGd involve caspase activation and provide a rationale to evaluate MGd in the treatment of lymphoma and leukemia patients.

Humanized Anti CD-40 Antibody SGN-40 Effectively Induces Cytotoxicity Against Chronic Lymphocytic Leukemia (CLL) Cells through Antibody Mediated Cytotoxicity and Demonstrates Modest Biologic Evidence of CD40 Activation.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2966-2966 ◽  
Author(s):  
Aruna C. Gowda ◽  
Xiaobin B. Zhao ◽  
Carolyn Cheney ◽  
Najma Mehter ◽  
Gerard Lozanski ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Cell Activation ◽  
Cd40 Ligand ◽  
Lymphocytic Leukemia ◽  
Extrinsic Pathway ◽  
Concurrent Administration ◽  
Cd40 Ligation ◽  
Cd40 Activation ◽  
Induced Apoptosis

Abstract The CD40 antigen is involved in cell survival and differentiation of B-cells and is uniformly expressed on chronic lymphocytic leukemia (CLL) cells. The CD40/CD40L interaction stimulates B-cells, dendritic cells and monocytes to proliferate, differentiate, up regulate co-stimulatory molecules and increase antigen presentation. While activation of CD40 can protect CLL cells against early fludarabine-induced apoptosis, these cells become sensitive to delayed death by extrinsic pathway apoptosis. (Blood, 105: 3193–8, 2005). SGN-40 is a humanized anti-CD40 antibody entering clinical trials and has been reported to have weak agonistic properties following CD40 ligation. To pursue rational clinical development of SGN-40, we studied the effects of this antibody in fresh, non-cryopreserved primary CLL cells. These studies included classic antibody mediated killing mechanisms and evidence of both CLL cell activation and protection against early fludarabine-mediated apoptosis. CLL cells treated with SGN-40 (10 mcg/ml) for 2 hours (hrs) in the presence of human serum promoted no complement mediated cytoxicity (CDC) in 8 pts tested. Direct SGN-40 induced apoptosis of human CLL cells with or without anti-Fc IgG cross-linking at 24, 48 and 72 hrs was not increased over that observed with the isotype control antibody trastuzumab in 8 pts studied. In contrast, SGN-40 induced antibody dependent cellular cytotoxicity (ADCC) against CLL cells an average of 12% (±11.39 SD, range 2–32%) killing at 4 hrs (effector to target cell ratio 25:1) in 6 pts tested. The SGN-40 induced ADCC against CLL cells were similar to that observed with alemtuzumab (average 19%, SD 6.9, range10–30%) or rituximab (average 18%, SD 12.48, range 8–42.5%). SGN-40 also mediated death in Raji and 697 lymphoblastic lymphoma cell lines via ADCC. Similar to reports by others with CD40 ligand, SGN-40 mediated activation was noted with modest up-regulation of CD80 and HLA-DR at 48hrs. When administered prior to fludarabine, SGN-40 also protected against death in 5 consecutive samples, although this was less than observed with CD40 ligand transfected HeLa cells, consistent with incomplete CD40 activation. Concurrent administration of SGN-40 and fludarabine did not protect from drug-mediated apoptosis. In conclusion, these findings suggest that SGN-40 has dual property of mediating cytotoxic effect by ADCC and partial CD40 activation. Development of SGN-40 as a therapeutic agent in CLL is justified and future studies by our group are focusing on enhancing SGN-40 mediated ADCC against CLL cells and potentially designing combination studies with SGN-40 to exploit this agent’s ability to engage the CD40/CD40L network.

Apoptosis in Response to Inhibition of Dipeptidyl Peptidase 2 (DPP2) Defines Low Risk Chronic Lymphocytic Leukemia (CLL).

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3094-3094
Author(s):  
Alexey V. Danilov ◽  
Olga V. Danilova ◽  
Andreas K. Klein ◽  
Brigitte T. Huber
Keyword(s):  
Cell Cycle ◽  
Cell Death ◽  
B Cells ◽  
Treatment Regimen ◽  
Protease Activity ◽  
Specific Inhibitor ◽  
Annexin V ◽  
Dipeptidyl Peptidase ◽  
Mrna Levels ◽  
Protein Levels

Abstract Maintenance of the G0 state is a key to survival of CLL B-cells. Heterogeneity of prognosis suggests that CLL is not a uniform disease. Molecules expressed in CLL with unfavorable prognosis, such as ZAP-70, Lyn, CD38 and others, provide stimuli which, coupled with B-cell receptor signaling, may alter cell cycle progression and delay apoptosis. We studied mechanisms of apoptosis in CLL B-cells via inhibition of Dipeptidyl Peptidase 2 (DPP2). DPP2 is a serine protease cloned in our lab, which is involved in the maintenance of the G0 (quiescent) state. Inhibition of DPP2 triggers apoptosis in healthy B-cells. 50 patients with B-CLL were recruited from the Hematology clinics at Tufts-NEMC (Boston, MA). Median time from diagnosis of CLL to enrollment in the study was 7 years with median follow up of 10.5 years. 24 patients (48%) have received treatment in the course of their disease. CLL B-cells were isolated from peripheral blood with standard Ficoll-Hypaque technique, treated with ValboroPro (Point Therapeutics), a non-specific inhibitor of DPPs, and/or AX8819 (ActivX), a DPP2-specific inhibitor, incubated for 16 hours and stained with anti-CD19 antibodies, propidium iodide and Annexin V. Expression of DPP2 and ZAP-70 mRNA was assessed by RT-qPCR, p27 protein - by western blot analysis. By blocking DPP2 protease activity, we distinguished two subsets of CLL - sensitive (S-CLL) and resistant (R-CLL) to DP-P2 inhibition-induced apoptosis. In 30 cases (60%), inhibition of DPP2 resulted in caspase-dependent apoptosis of CLL B-cells (S-CLL). 70–90% of B-cells stained Annexin V-positive after incubation with AX8819. Pre-incubation with Rituximab (Biogen Idec) did not enhance cell death. In the remaining 20 cases (40%) inhibition of DPP2 did not cause cell death (R-CLL). R-CLL demonstrated higher expression of ZAP-70 mRNA (p<0.001) and lower levels of p27 protein (p<0.01) than S-CLL. ZAP-70 mRNA levels strongly correlated with resistance to apoptosis (χ2=26.7, p<0.0001). Inhibition of DPP2 resulted in a decrease in p27 protein levels in S-CLL, but not in R-CLL. Both groups expressed higher DPP2 mRNA levels than B-cells derived from healthy donors. In the R-CLL subgroup, 18 patients (84%) required treatment for their disease, 14 received more than one treatment regimen, 2 underwent allogeneic transplant and 2 died of complications of CLL. Among the S-CLL cohort, 10 patients (33%) initiated treatment, 4 received more than one treatment regimen. R-CLL cohort required treatment earlier than S-CLL (3.5 vs. 9.9 years from diagnosis). Thus, DPP2 inhibition discriminates two subsets of CLL based on their ability to undergo apoptosis upon disruption of the quiescent program. We postulate that DPP2 is critical for the maintenance of G0 in S-CLL. Its inhibition leads to inappropriate cell cycle entry, as evidenced by a decrease in p27 protein levels and cell death. The R-CLL subgroup expresses high ZAP-70 mRNA levels which portends a worse clinical course. In this subgroup, ZAP-70 kinase activity may provide an additional tonic signal that pushes CLL B-cells into late G0/early G1. At this stage, DPP2 protease activity is no longer required for survival. DPP2 inhibition may serve as an easy-to-perform prognostic test, as well as a novel therapeutic approach.

Lenalidomide-Dependent Activation of the Phosphatidylinositol 3-kinase-δ Pathway Is Antagonized by CAL-101 In Chronic Lymphocytic Leukemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1821-1821
Author(s):  
Sarah E.M. Herman ◽  
Rosa Lapalombella ◽  
Jeffrey A. Jones ◽  
Leslie Andritsos ◽  
Amber L. Gordon ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Research Funding ◽  
Cell Activation ◽  
Specific Inhibitor ◽  
Lymphocytic Leukemia ◽  
Clinical Activity ◽  
Cytokine Release ◽  
Treatment Naïve ◽  
Previously Treated

Abstract Abstract 1821 Lenalidomide is an oral immune-modulating agent that has been shown to have clinical activity in patients with treatment-naive and previously treated chronic lymphocytic leukemia (CLL). In CLL, a disease-specific phenomenon of drug-induced tumor flare is often observed that results in lymph node enlargement, rash, and cytokine release. We and others have attributed both lenalidomide-induced tumor flare and cytokine release in part to CLL cell activation, with concomitant increase in surface co-stimulatory molecules including CD154. The potential consequences of such activation by lenalidomide in CLL are multiple. In symptomatic, previously untreated CLL, activation of tumor cells by lenalidomide likely contributes to reversal of hypogammaglobulinemia in a subset of patients. Additionally, activation of CLL cells increases their capacity for antigen presentation, potentially facilitating a clinically beneficial development of tumor-specific antibodies toward antigens such as ROR1. In patients with previously treated CLL, lenalidomide therapy does not reverse hypogammaglobulinemia. However, treatment has been documented to increase serum b-FGF and VEGF levels, which correlates with lack of response. Previous work demonstrates that CLL cells predominately utilize the PI3K p110δ isoform for activation following CD154 signaling. Given our prior findings of prominent lenalidomide induction of CD40-CD154 signaling in vitro and in vivo, we focused initially on molecular interrogation of isoforms responsible for this in CLL cells. Utilizing primary CLL cells, we demonstrated that inhibition of PI3K-δ signaling by CAL-101, a clinically relevant PI3K-δ isoform-specific inhibitor, abrogated lenalidomide-induced activation of CLL cells by directly reducing PI3K enzymatic activity and also reducing phosphorylation of the downstream PI3K target AKT. Parallel studies with siRNA targeted to the p110δ isoform of PI3K demonstrated antagonism of lenalidomide-induced AKT phosphorylation. Furthermore, we found that inhibition of PI3K-δ by CAL-101 at therapeutically relevant concentrations (1 μM) prevented up-regulation of CD40, CD154, and CD86 by lenalidomide and also antagonized production of IgM by normal B-cells co-cultured with CLL cells. Collectively, these data demonstrate the importance of PI3K-δ signaling in modulating the pharmacological effects of lenalidomide in CLL cell activation including up-regulation of CD40, CD154, CD86 and active CLL cell co-stimulation of normal B-cells. Our findings suggest that clinical evaluation of combination strategies of lenalidomide and CAL-101 in treatment-naive patients with CLL should be performed with careful pharmacodynamic monitoring of immune modulation and signaling to best preserve the clinical benefits of both drugs. This work is supported by the Leukemia and Lymphoma Society, D. Warren Brown Foundation, and The OSU Leukemia SPORE grant funded by the NCI. CAL-101 was provided by Calistoga Pharmaceuticals, Inc. Disclosures: Jones: Glaxo Smith-Kline: Consultancy; Abbott: Research Funding. Lannutti:Calistoga Pharmaceutical Inc.: Employment. Byrd:Calistoga Pharmaceutical Inc.: Equity Ownership. Johnson:Calistoga Pharmaceutical Inc.: Research Funding.

scholarly journals CD5-induced apoptosis of B cells in some patients with chronic lymphocytic leukemia

Leukemia ◽  
2002 ◽  
Vol 16 (1) ◽  
pp. 44-52 ◽  
Author(s):  
JO Pers ◽  
C Berthou ◽  
N Porakishvili ◽  
M Burdjanadze ◽  
G Le Calvez ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Lymphocytic Leukemia ◽  
Induced Apoptosis

scholarly journals Targeting Maternal Embryonic Leucine Zipper Kinase with OTSSP167 Displays Anti-Tumor Activities in Chronic Lymphocytic Leukemia through Down-Regulation of FoxM1

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 801-801
Author(s):  
Ya Zhang ◽  
Xiangxiang Zhou ◽  
Ying Li ◽  
Yangyang Xu ◽  
Xin Wang
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Cell Proliferation ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Leucine Zipper ◽  
Lymphocytic Leukemia ◽  
Functional Enrichment ◽  
Down Regulation ◽  
Cell Chemotaxis

Abstract Introduction Maternal embryonic leucine zipper kinase (MELK) is a novel oncogene, which exerts pivotal roles in maintenance of cancer stem cells. OTSSP167, an orally bioavailable inhibitor targeting MELK, is currently in a phase I/II clinical trial in patients with acute myeloid leukemia and breast cancer. Yet, no literature has been reported regarding the effects of MELK and OTSSP167 in chronic lymphocytic leukemia (CLL). Intriguingly, previous studies identified that OTSSP167 contributed to tumorigenesis via p53 pathway, highlighting its potential efficacy in relapsed/ refractory CLL. Hence, the aim of this study was to determine the anti-tumor potency and mechanism of OTSSP167 targeting MELK in CLL. Methods Peripheral blood samples from 55 de novo CLL patients were collected with informed consents at the Department of Hematology in Shandong Provincial Hospital Affiliated to Shandong University (SPHASU). CD19+ B cells were isolated with informed consents from healthy donors. Expression levels of MELK mRNA and protein in CLL cells were determined by quantitative RT-PCR and western blotting. Kaplan-Meier survival curves with log-rank test of overall survival (OS) were analyzed. Microarray datasets GES22762 and GSE25571 were obtained from Gene Expression Omnibus. Functional enrichment analyses of gene ontology (GO), kyoto encyclopedia of genes and genomes (KEGG) and gene set enrichment analysis (GSEA) in gene expression profiles were performed. Lentiviral vectors transfected CLL cells to stably silence MELK and adenovirus delivery were constructed to overexpress MELK. Besides, cell viability, apoptosis and cell cycle were assessed by cell counting kit-8, annexin V-PE/7AAD and PI/RNase staining, respectively. Cell migration was evaluated by CXCL12-induced chemotaxis. Results Aberrantly increased expression of MELK was detected in CLL primary cells and cell lines (MEC1 and EHEB) at mRNA and protein level compared with normal B cells (Figure 1A-D). We observed MELK over-expression in significant correlation with higher WBC count( p=0.022), advanced Binet stage (p=0.023), high-risk Rai stage (p=0.022), elevated LDH level (p=0.002) and increased β2-MG levels (p=0.009), unmutated IGHV (p=0.037), positive ZAP-70 (p=0.037) and deletion of 17p13 (p=0.009). Besides, MELK high expression was revealed in significant association with reduced OS of enrolled patients, which was further confirmed in GSE22762 (Figure 1E). Annotations of bioinformatics analyses indicated that MELK was functional enriched in cell proliferation, regulation of G2/M cell cycle, response to drug and p53 signaling pathway in cancer. In consistent with functional enrichment analyses, CLL cells with silence or inhibition of MELK exhibited attenuated cell proliferation, increased fast-onset apoptosis, induced G2/M phase arrest, impaired cell chemotaxis and enhanced sensitivity to fludarabine and ibrutinib (Figure 2A-E). Whereas, gain-of-function assay showed promoted cell proliferation and cell chemotaxis (Figure 2E-F). Additionally, serial dilution of OTSSP167 decreased phosphorylation of AKT and ERK1/2 in CLL cells (Figure 3A). Besides, expression of phosphorylated FoxM1, FoxM1, cyclinB1 and CDK1 decreased with treatment of OTSSP167 in CLL cells. p53 modestly changed with MELK suppression in MEC1 cells, yet visibly elevated in EHEB cells. Besides, activation of p21 was observed (Figure 3B). Furthermore, confocal immunofluorescent images revealed that MELK and FoxM1 co-localized in CLL cells and were down-regulated by OTSSP167 in a dose-dependent manner (Figure 3C). Collectively, the interactive network of MELK and FoxM1-signaling cascade in genomic expression profile was established, illuminating the potential mechanism of MELK inhibition by OTSSP167 in CLL (Figure 3D). Conclusion Our investigations identified for the first time the oncogenic role of MELK in CLL tumorigenesis and regulatory mechanism of OTSSP167 in CLL cells by bioinformatics analysis and ex vivo evaluation. Expression of MELK was upregulated, and associated with adverse outcome of CLL patients. OTSSP167 exerted therapeutic potential at nanomolar concentrations in abrogating cell survival and migration, inducing cell cycle arrest through down-regulation of FoxM1. Further clinical investigation is warranted to substantiate OTSSP167 as a novel therapeutic strategy in progressed CLL. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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