BCL-2 Phosphorylation Modulates Sensitivity to the BH3-Mimetic GX15-070 (Obatoclax) and Reduces Its Synergistic Interaction with Bortezomib in Chronic Lymphocytic Leukemia Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3464-3464 ◽  
Author(s):  
Patricia Perez Galan ◽  
Gael Roue ◽  
Monica Lopez Guerra ◽  
Neus Villamor ◽  
Emili Montserrat ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Ex Vivo ◽  
Single Agent ◽  
Proteasome Inhibition ◽  
Hematologic Malignancies ◽  
Lymphocytic Leukemia ◽  
Primary Cells ◽  
Apoptotic Pathway ◽  
Bh3 Mimetic

Abstract Chronic lymphocytic leukemia (CLL) is the most common leukemia in Western countries and is characterized by the accumulation of CD5-positive monoclonal B cells. This clonal excess of B cells is caused by a concomitant defect in both cell death and proliferation. A key factor that explains this inappropriate cell survival is the imbalanced expression of BCL-2 family proteins, thus representing an attractive therapeutic target for the treatment of this neoplasm. The current strategies for BCL-2 antagonism are based on small molecules that target several antiapoptotic BCL-2 proteins by mimicking a BH3 domain. Among them, GX15-070/Obatoclax (GeminX Biotechnologies) is pan-BCL-2 inhibitor that binds to BCL-2, BCL-W, BCL-XL and MCL-1 with high affinity, and has shown efficacy against several hematologic malignancies and solid tumors. In the present work, we report how GX15-070 led to the disruption of BCL-2/BIM and MCL-1/BAK complexes in CLL cells after short incubation times (3h), followed by the activation of the mitochondrial apoptotic pathway. Ex vivo experiments in CLL primary cells showed that GX15-070 as a single agent induces apoptosis at pharmacological concentrations. GX15-070 is also effective in CLL cells presenting alterations in P53, ATM, 13q deletions or high levels of ZAP-70 expression. LD50 at 20h were significantly higher in CLL cells (5.95 + 2.8 μM) compared to those previously reported in mantle cell lymphoma (MCL) primary cells (2.93 + 2.48 μM) (P<0.01). Of interest, these differences correlated with higher levels of pBCL-2(Ser70) in CLL compared to MCL primary cells. In the same context, we also demonstrated that ZAP-70+ CLL cases, which showed higher LD50 values than ZAP-70- ones, also expressed higher levels of pBCL-2(Ser70). Considering that BCL-2 phosphorylation at serine 70 residue is required for its antiapoptotic function, and that limits its interaction with proapoptotic multidomain and BH3-only proteins, it is conceivable that high levels of phosphorylated BCL-2 could impede or reduce GX15-070 activity. Both ERK1 (p44) and ERK2 (p42) kinases have been proposed to be responsible for BCL-2 phosphorylation. Considering these studies, we have demonstrated that pharmacological inhibition of MEK1/ERK pathway by PD98059 is able to reduce pBCL-2(Ser70) levels, increasing GX15-070 activity in CLL primary cells. In addition, as the protein phosphatase PP2A has been found to be responsible for BCL-2 dephosphorylation, its inhibition by okadaic acid increased pBCL-2(Ser70) levels, reducing GX15-070 cytotoxic activity. GX15-070 activity was increased by cotreatment with the proteasome inhibitor bortezomib. However, as proteasome inhibition led to the accumulation of pBCL-2(Ser70), the degree of interaction between GX15-070 and bortezomib was also regulated by the levels of pBCL-2(Ser70). Accordingly, as ERK1/2 is responsible for this phosphorylation, we also demonstrated that ERK1/2 inhibition by PD98059 could reverse bortezomib-induced accumulation of pBCL-2(Ser70) and increased GX15-070 and bortezomib cytotoxic effect. These results support the role of BCL-2 phosphorylation as a mechanism of resistance to BH3 mimetic compounds, and demonstrate that combination approaches including ERK inhibitors could enhance BH3 mimetics activity both alone or in combination with proteasome inhibitors.

The Role of Proteasome Inhibitors and the Trail Apoptotic Pathway in the Treatment of Chronic Lymphocytic Leukemia.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5011-5011
Author(s):  
Kristin E. McCrea ◽  
Albert Kabore ◽  
James B. Johnston ◽  
Spencer B. Gibson
Keyword(s):  
Multiple Myeloma ◽  
Monoclonal Antibodies ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Proteasome Inhibitor ◽  
Proteasome Inhibitors ◽  
Death Receptors ◽  
Lymphocytic Leukemia ◽  
Apoptotic Pathway ◽  
Novel Approach

Abstract TRAIL (tumour necrosis factor-related apoptosis-inducing ligand) triggers the TRAIL apoptotic pathway selectively in tumour cells by binding to two death receptors, DR4 and DR5, that are present on the surface of many cancer cells. It is effective at inducing apoptosis in a variety of haematological malignancies, such as multiple myeloma. However, chronic lymphocytic leukemia (CLL) cells are relatively resistant to TRAIL-induced apoptosis. We have previously shown that the chemotherapeutic drugs, fludarabine and chlorambucil, increase the cell surface expressions of DR4 and DR5, and give synergistic apoptotic responses when combined with TRAIL. Proteasome inhibitors, that are used in the treatment of multiple myeloma, also upregulate TRAIL and its death receptors in CLL cells but not in normal B cells. Herein we have determined that proteasome inhibitors are effective at inducing apoptosis in CLL cells, and that the activation of the TRAIL apoptotic pathway contributes significantly to proteasome inhibitor cytotoxicity. Combining proteasome inhibitors with TRAIL enhanced apoptosis in CLL cells by approximately 15% over treatment with the proteasome inhibitor alone. Another novel approach to trigger the TRAIL apoptotic pathway is to use activating monoclonal antibodies directed against DR4 and DR5. Similar to TRAIL, we demonstrated that when used alone, the monoclonal antibodies were minimally cytotoxic against CLL cells. Proteasome inhibitors in combination with activating monoclonal antibodies against DR4 or DR5 increased the amount of apoptosis in CLL cells. Cell death was enhanced by 23% and 17% when proteasome inhibitors were combined with monoclonal antibodies against DR4 and DR5, respectively. While proteasome inhibitors may have a potential role in CLL treatment as single therapy, they are also cytotoxic to normal B cells. However, when activating monoclonal antibodies against TRAIL death receptors are given in combination with proteasome inhibitors, that upregulate DR4 and DR5 expression, the anti-tumor specificity and cytotoxicity of these agents may be increased in CLL.

IL-21 Promotes Apoptosis through Induction of the BH3 Only Protein Bim and Enhacnes Direct and Innate Immune-Promoted Death of Chronic Lymphocytic Leukemia Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3118-3118
Author(s):  
Julie M. Roda ◽  
Aruna Gowda ◽  
Rehan Hussain ◽  
Asha Ramanunni ◽  
Amy Lehman ◽  
...  
Keyword(s):  
Clinical Trials ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Nk Cell ◽  
Single Agent ◽  
Innate Immune ◽  
Lymphocytic Leukemia ◽  
Clinical Activity ◽  
Specific Lysis

Abstract Chemoimmunotherapy with fludarabine and the anti-CD20 monoclonal antibody rituximab has demonstrated promising clinical activity in chronic lymphocytic leukemia (CLL). We hypothesized that the gamma chain receptor cytokine IL-21, which is currently in clinical trials for lymphoma, might enhance the efficacy of this regimen by augmenting both immune-mediated clearance of CLL cells and a direct apoptotic mechanism involved in the homeostasis of normal B cells. CLL cells expressed variable levels of the IL-21 receptor alpha subunit (<10–80% positive cells, n = 16). IL-21 induced direct apoptosis in CLL cells from a subset of patients (40% ± 15.9 apoptotic cells; n = 9; p <0.0001), which directly correlated with increased expression of surface IL-21R (p = 0.001). The in vitro apoptosis occurred at physiologically attainable concentrations (25 ng/ml) and was time- and dose-dependent. As a single agent, IL-21 did not activate CLL cells, as evidenced by lack of surface expression of CD86, HLADR, CD95, or CD40. However, IL-21 induced phosphorylation of STAT-1Tyr-701 and STAT-3 Tyr-705 in CLL cells exhibiting > 20% apoptosis at 72 hours, whereas phosphorylation of these proteins was not seen in CLL cells failing to undergo apoptosis. Similar to normal murine B cells, IL-21-mediated death was associated with up-regulation of the pro-apoptotic BH3 only domain protein Bim, whereas Bim was not up-regulated in CLL cells insensitive to IL-21-induced apoptosis. Furthermore, silencing of Bim in primary CLL cells with siRNA antagonized IL-21-mediated death. Preliminary studies examining the mechanism of Bim up-regulation demonstrated that both total levels and phosphorylation of FOXO3AThr32 increases following IL-21 treatment. FOXO3a is involved in transcriptional regulation of Bim, and further mechanistic studies are ongoing and will be presented. Given the favorable modulation of Bim, we examined the ability of IL-21 to enhance apoptosis in response to rituximab, alemtuzumab, or fludarabine. Our studies confirm that CLL cells pre-treated with IL-21 are sensitized to rituximab and fludarabine, whereas IL-21 had no effect on fludarabine-mediated apoptosis of normal T cells. IL-21 also enhanced NK cell ADCC against rituximab-coated autologous CLL cells (42 ± 4.4% rituximab-specific lysis vs. 28 ± 3.1% at an E:T ratio of 25:1; p < 0.0001). These data provide evidence that IL-21 promotes direct apoptosis through induction of Bim and also enhances fludarabine- and rituximab-mediated apoptosis. Additionally, IL-21 enhances autologous innate immune activation of NK cells toward primary CLL cells coated with rituximab. Overall, these findings provide justification for combination studies of IL-21 with fludarabine and rituximab chemoimmunotherapy in CLL and point to Bim induction as a pharmacodynamic endpoint to predicting surrogate biologic activity of IL-21 in vivo as part of planned clinical trials with this agent.

Targeting Early Events of B-Cell Receptor Signaling in Chronic Lymphocytic Leukemia: Suppressed Syk and PLCγ2 Activities Predict Apoptotic Response of Leukemic Cells to Dasatinib

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5023-5023
Author(s):  
Y. Lynn Wang ◽  
Zibo Song ◽  
Pin Lu ◽  
John P. Leonard ◽  
Morton Coleman ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Kinase Inhibitor ◽  
Ex Vivo ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
Bcr Signaling

Abstract B cell receptor (BCR) signaling plays an essential role in the pathogenesis of chronic lymphocytic leukemia. In a subset of patients with a poor clinical outcome, BCR ligation leads to increased cell metabolism and cell survival (Cancer Research66, 7158–66, 2006). Based on these findings, we tested whether targeting BCR signaling with dasatinib, an inhibitor of Src kinase, would interfere with the signaling cascade and cause death of CLL B cells. CLL leukemic cells were isolated from 34 patients and were incubated with or without dasatinib at a low dose of 128 nM. Among 34 cases, viability of leukemic cells was reduced by 2% to 90%, with an average of ~50% reduction on day 4 of ex vivo culture. Further study showed that CLL B cells undergo death by apoptosis via the intrinsic pathway which involves the generation of reactive oxygen species. Analysis of the Src family kinases showed that phosphorylation of Src, Lyn and Hck was inhibited by dasatinib not only in those cases that responded to dasatinib with apoptosis, but also in those that did not respond well (&lt;20% apoptosis). Further analysis revealed that suppressed activity of two downstream molecules, Syk and PLC Statistical analysis showed a significant correlation between CLL dasatinib response and their IgVH mutation and ZAP70 status. Cases with worse prognoses by these criteria have a better response to the kinase inhibitor. Lastly, we have also found that ZAP70 positive cases showed a greater degree of PLC

Efficient Gene Delivery to Chronic Lymphocytic Leukemia (CLL) Cells with Microbubbles Bearing ROR1 Antibody

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2857-2857
Author(s):  
Suping Zhang ◽  
Wenjin Cui ◽  
Robert Mattrey ◽  
Thomas J. Kipps
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Duty Cycle ◽  
Research Funding ◽  
Ex Vivo ◽  
Lymphocytic Leukemia ◽  
Ultrasound Treatment ◽  
Targeted Microbubbles ◽  
Receive Treatment

Abstract Abstract 2857 Patients of chronic lymphocytic leukemia (CLL) typically develop progressive immune deficiency, which impairs their response to vaccines. Prior studies showed that infusions of autologous CLL cells transduced ex vivo with adenovirus encoding CD154 could elicit anti-leukemia immune responses. However, the need for high amounts of high-titer adenovirus complicates this approach and handicaps clinical development. A recently developed technology, known as microbubbles, could be activated using safe ultrasound (U/S) energy, potentially providing a new tool with which to affect gene delivery. In this study, we examined whether microbubble-technology could effectively transfect CLL B cells ex vivo or in vivo. To target CLL cells with microbubbles, we used newly generated mAb specific for ROR1, a surface antigen expressed on CLL B cells, but not on normal B cells or most other adult tissues. Anti-ROR1 mAb was incorporated into the lipid shell of microbubble, which was composed with DSPC and DSPE-PEG2000-Maleimide via maleimide chemistry. We examined whether targeting the microbubbles with anti-ROR1 mAb improved our transfection efficiency for ROR1+ CLL cells. For this we generated microbubbles tagged with anti-ROR1 mAb or control Ig and incubated these with fresh whole blood from patients with CLL. We found that targeted bubbles specifically bound to CD19+CD5+ CLL cells, but not to other cells, including RBCs. Next we examined whether anti-ROR1-tagged microbubbles or non-targeted microbubbles could transfect CLL cells with an expression plasmid (pGFP) encoding green-fluorescence protein (GFP). Isolated CLL cells were incubated with pGFP-laden microbubbles for 1 hour at 37° C. Following this the preparations were exposed to ultrasound at 2w/cm2 and 50% duty cycle or left untreated. After 48 hours culture, the cells were collected and stained with propidium iodide (PI) and examined via flow cytometry. Using these conditions, CLL cells in all treatment groups retained high viability (>80%). CLL cells incubated with anti-ROR1 mAb-tagged microbubbles expressed high levels of GFP provided they had also been exposed to ultrasound; CLL cells treated with anti-ROR1-mAb-tagged microbubbles that did not receive ultrasound treatment remained negative for expression of GFP. Similarly, CLL cells treated with non-targeted pGFP-laden microbubbles did not express GFP regardless of whether they also received ultrasound treatment. We also examined whether anti-ROR1-mAb-tagged microbubbles could transfect CLL cells in vivo. For this 107 human CLL B cells were injected into the peritoneum of each RAG-2−/−/ γc−/− mouse. Five minutes later, anti-ROR1-mAb-tagged microbubbles or non-targeted microbubbles laden with expression vectors encoding GFP and luciferase were injected into the peritoneum of each animal. Ten minutes later groups of animals did or did not receive treatment with ultrasound at 2w/cm2 and 50% duty cycle for 1 min. Forty-eight hours later mice were examined for reporter gene expression via whole body imaging. The group of animals that received anti-ROR1-mAb-tagged microbubbles and ultrasound treatment all had high-level expression of luciferase. Cells were recovered from the mice via peritoneal lavage. Immunofluorescence studies confirmed that only ROR1+CD5+ cells expressed GFP. In contrast mice injected with anti-ROR1-mAb-tagged microbubbles but did not receive treatment with ultrasound had no detectable luciferase activity. Groups of mice that were treated with non-targeted microbubbles also had negligible leuciferase activity regardless of whether they received ultrasound treatment. This study reveals that anti-ROR1-mAb tagged microbubbles that are activated by extracorporeal ultrasound treatment can be effective vehicles for specific delivery of genes into CLL cells ex vivo and in vivo. Disclosures: Kipps: Igenica: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Research Funding; Abbott Industries: Research Funding; Genentech: Research Funding; GSK: Research Funding; Gilead Sciences: Consultancy, Research Funding; Amgen: Research Funding.

Forodesine has high antitumor activity in chronic lymphocytic leukemia and activates p53-independent mitochondrial apoptosis by induction of p73 and BIM

Blood ◽  
2009 ◽  
Vol 114 (8) ◽  
pp. 1563-1575 ◽  
Author(s):  
Roberto Alonso ◽  
Mónica López-Guerra ◽  
Ramanda Upshaw ◽  
Shanta Bantia ◽  
Caroline Smal ◽  
...  
Keyword(s):  
Dna Damage ◽  
Chronic Lymphocytic Leukemia ◽  
Clinical Investigation ◽  
Single Agent ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
Apoptotic Pathway ◽  
Incurable Disease ◽  
Chemotherapeutic Approach ◽  
High Antitumor Activity

Abstract Chronic lymphocytic leukemia (CLL) is an incurable disease derived from the monoclonal expansion of CD5+ B lymphocytes. High expression levels of ZAP-70 or CD38 and deletions of 17p13 (TP53) and 11q22-q23 (ATM) are associated with poorer overall survival and shorter time to disease progression. DNA damage and p53 play a pivotal role in apoptosis induction in response to conventional chemotherapy, because deletions of ATM or p53 identify CLL patients with resistance to treatment. Forodesine is a transition-state inhibitor of the purine nucleoside phosphorylase with antileukemic activity. We show that forodesine is highly cytotoxic as single agent or in combination with bendamustine and rituximab in primary leukemic cells from CLL patients regardless of CD38/ZAP-70 expression and p53 or ATM deletion. Forodesine activates the mitochondrial apoptotic pathway by decreasing the levels of antiapoptotic MCL-1 protein and induction of proapoptotic BIM protein. Forodesine induces transcriptional up-regulation of p73, a p53-related protein able to overcome the resistance to apoptosis of CLL cells lacking functional p53. Remarkably, no differences in these apoptotic markers were observed based on p53 or ATM status. In conclusion, forodesine induces apoptosis of CLL cells bypassing the DNA-damage/ATM/p53 pathway and might represent a novel chemotherapeutic approach that deserves clinical investigation.

Patterns of Venetoclax Sensitivity in Chronic Lymphocytic Leukemia

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 12-14
Author(s):  
James T Dibb ◽  
Nicola Long ◽  
Christopher A. Eide ◽  
Stephen E Kurtz ◽  
Cristina E. Tognon ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Board Of Directors ◽  
Research Funding ◽  
Ex Vivo ◽  
Single Agent ◽  
Drug Combinations ◽  
Lymphocytic Leukemia ◽  
Publicly Traded ◽  
Private Company ◽  
Advisory Committees

Patterns of Venetoclax Sensitivity in Chronic Lymphocytic Leukemia Chronic lymphocytic leukemia (CLL) is predominantly a disease of older adults. The 5-year overall survival is 70-91%, depending on Rai/Binet stage at diagnosis (80% overall), and although a subset of CLL takes a very indolent course, it can be aggressive as well. Disease course and responsiveness to therapeutic agents may be predictable, to some degree, based on specific genetic lesions or other patient population characteristics. Monotherapies targeting specific cell pathways are rapidly increasing in prevalence. Ibrutinib (Bruton tyrosine kinase inhibitor) has shown promise as a single agent as well as in combination with other agents. In particular, ibrutinib has shown efficacy in combination with venetoclax (inhibitor of cell death suppressor BCL2). This combination appears to be particularly potent in patients with a del(11q) karyotype. Cytogenetic information is used already in several other leukemias to inform prognosis and treatment. Although CLL is a disease of monoclonal proliferation, precise definition of the diseased clone will allow for more individualized treatment. Stratification of drug sensitivity based on genetic and cytogenetic features will directly affect patient outcomes in CLL. Primary patient mononuclear cells (from either peripheral blood or bone marrow) were plated ex vivo with a panel of 49 drug combinations and the 16 respective single agents (SA) in 384-well plates using 10,000 cells/well. Drugs were tested in 7-point concentration series; wells with drug combinations were added at fixed molar ratios. Cell viability was assessed after a 72 hour culture period. In this assay, primary cells maintain viability but do not proliferate. In CLL, the most frequent mutations were: del(17p); del(11q); del(13q14); trisomy 12; complex karyotype (at least three chromosomal aberrations). Selected analysis of these data from 157 unique patients were performed by isolating the most potent inhibitors (defined by lowest median AUC) either as a single agent or in combination with known treatments. These were evaluated with nonparametric tests (Kruskal-Wallace, Mann-Whitney, Spearman rank coefficient) on the statistical software Prism. By subdividing the data by available genetic and cytogenetic information, patterns that have not been previously described in the literature emerged. In the cohort of patients with any karyotypic abnormality (not complex karyotype), SA venetoclax and the combination of venetoclax-ibrutinib (VEN/IBRUT) were equivalently effective with no significant difference in efficacy observed between SA venetoclax and the combination. As previously described, del(11q) independently predicts increased efficacy of SA venetoclax and VEN/IBRUT, and this efficacy was validated by ex vivo potency here as well. However, we show that male gender is an independent predictor of potency in both SA venetoclax and VEN/IBRUT as well. Interestingly, doramapimod (an inhibitor of p38 MAP kinase) was not particularly potent as a SA, however, the combination of venetoclax-doramapimod (VEN/DORA) proved to be the most potent of all combinations tested, more potent than even VEN/IBRUT. This effect could not be replicated in any subgroup, as VEN/DORA samples for the entire cohort were relatively limited (n=31). Although this analysis has inherent limitations, including underpowered data to analyze in less frequent cytogenetic events (e.g. del(6q)), we did find significant patterns of potency. These may or may not translate to clinical efficacy in CLL and do not address any potential toxicity. However, these data suggest future directions for more targeted research on these drugs and drug combinations. Disclosures Tyner: Petra:Research Funding;Janssen:Research Funding;Seattle Genetics:Research Funding;Incyte:Research Funding;Genentech:Research Funding;Constellation:Research Funding;AstraZeneca:Research Funding;Aptose:Research Funding;Gilead:Research Funding;Takeda:Research Funding;Syros:Research Funding;Agios:Research Funding;Array:Research Funding.Druker:EnLiven:Consultancy, Research Funding;Gilead Sciences:Consultancy, Membership on an entity's Board of Directors or advisory committees;Cepheid:Consultancy, Membership on an entity's Board of Directors or advisory committees;Dana-Farber Cancer Institute:Patents & Royalties;Bristol-Myers Squibb:Research Funding;Blueprint Medicines:Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees;Aptose Therapeutics Inc. (formerly Lorus):Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees;ARIAD:Research Funding;Third Coast Therapeutics:Membership on an entity's Board of Directors or advisory committees;The RUNX1 Research Program:Membership on an entity's Board of Directors or advisory committees;Pfizer:Research Funding;Patient True Talks:Consultancy;Oregon Health & Science University:Patents & Royalties;Novartis Pharmaceuticals:Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding;MolecularMD (acquired by ICON):Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees;Millipore (formerly Upstate Biotechnology):Patents & Royalties;VB Therapeutics:Membership on an entity's Board of Directors or advisory committees;Vivid Biosciences:Membership on an entity's Board of Directors or advisory committees;ALLCRON:Consultancy, Membership on an entity's Board of Directors or advisory committees;Amgen:Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees;Aileron Therapeutics:Membership on an entity's Board of Directors or advisory committees;Merck & Co:Patents & Royalties;McGraw Hill:Patents & Royalties;GRAIL:Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees;Henry Stewart Talks:Patents & Royalties;Iterion Therapeutics (formerly Beta Cat Pharmaceuticals):Membership on an entity's Board of Directors or advisory committees;Leukemia & Lymphoma Society:Research Funding.

Triterpenoids Display Single Agent Activity in a Mouse Model of CLL/SBL.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2530-2530
Author(s):  
Christina L. Kress ◽  
Marina Konopleva ◽  
Maryla Krajewska ◽  
Vanesa Martinez-Garcia ◽  
Sophie Lefebvre ◽  
...  
Keyword(s):  
B Cells ◽  
Mouse Model ◽  
Drug Testing ◽  
Ex Vivo ◽  
Single Agent ◽  
B Cell Lymphoma ◽  
Lymphocytic Leukemia ◽  
Clinical Activity ◽  
Potent Inducer

Abstract The synthetic triterpenoid 2-Cyano-3,12-Dioxooleana-1,9-Dien-28-Oic Acid (CDDO) induces apoptosis of leukemia cells through a novel mechanism and has recently entered Phase I human clinical trials. We studied the effects of CDDO and its imidazolide derivative (CDDO-Im) on cultured human chronic lymphocytic leukemia (CLL) cells and on B cells from TRAF2DN/Bcl-2 transgenic mice, a new mouse model of CLL and small B cell lymphoma (SBL). Both triterpenoids efficiently induced death of malignant human and mouse B-cells ex vivo, although CDDO-Im was over 10-fold more potent than CDDO. Treating TRAF2DN/Bcl-2 mice that had developed leukemia with liposome-formulated CDDO or CDDO-Im resulted in significant reductions of B cells in blood, spleen and lung, with CDDO-Im more potent than CDDO, while treatment with empty liposomes had no impact on disease. Analysis of blood cells recovered from treated mice showed that CDDO-Im is a potent inducer of cell dead in vivo. Furthermore, CDDO-Im efficiently eradicated mouse CLL cells but had a lesser effect on the viability of normal B cells. These results demonstrate that triterpenoids CDDO and CDDO-Im reduce leukemia and lymphoma burden in vivo in a transgenic mouse model of CLL/SBL and suggest that CDDO-based synthetic triterpenoids should be tested for clinical activity in patients with CLL. Our results also provide evidence of the suitability of our mouse model of CLL/SBL as a preclinical platform for chemotherapeutic drug testing.

Incidence Of Atypical Chronic Lymphocytic Leukemia In 1819 Patients With B Chronic Lymphoproliferative Disorder

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1770-1770
Author(s):  
Edouard Cornet ◽  
Alice-Sophie Boucher ◽  
Véronique Salaun ◽  
Florence Truquet ◽  
Michele Malet ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Lymphoproliferative Disorder ◽  
Who Classification ◽  
Cell Lymphoma ◽  
Hematologic Malignancies ◽  
Lymphocytic Leukemia ◽  
Histological Evidence

Abstract Flow cytometry is the diagnostic tool of choice to study abnormal lymphoid population detected in peripheral blood by morphological analysis. The main diagnosed chronic lymphoproliferative disorder (CLPD) is chronic lymphocytic leukemia (CLL). In a significant number of cases, a B-CLPD non-CLL can be diagnosed. Further molecular and histological examinations are then compulsory to characterize such hematologic malignancies. The objective of this study was to determine the incidence of atypical CLL among all B-CLPD diagnosed by flow cytometry. We retrospectively studied the B-CLPD consecutively diagnosed at the hospital of Caen (Normandy, France) between 2000 and 2013. The diagnosis of B-CLPD was based on the detection by flow cytometry of circulating lymphoid abnormal B cells. Multiparametric flow cytometry included markers CD19, CD20, CD22, CD79b, CD5, CD10, CD23, CD43, FMC7, CD38 and light chains (kappa and lambda) of surface immunoglobulin. The diagnosis of CLL was based on the criteria defined by Hallek et al (Blood 2008). The non-CLL B-CLPD were then explored by molecular analyses driven by the phenotype of B-cells (overexpression of cyclin D1 in case of CD5+/CD10-/CD23- B-CLPD and BCL2-JH rearrangement in case of CD10+ B-CLPD). In addition, histological evidence was necessary to classify the B-CLPD non-CLL. 1819 B-CLPD were detected by flow cytometry. The distribution of B-CLPD was as follows: 1156 cases (64%) of CLL or immunophenotypic equivalent (leukemic phase of small lymphocytic lymphoma (SLL) and monoclonal B-cell lymphocytosis (MBL)), 297 cases (16%) of marginal zone lymphoma (MZL), 84 cases (5%) of mantle cell lymphoma (MCL), 39 cases (2%) of follicular lymphoma (FL), 26 cases (1%) of hairy cell leukemia (HCL), 13 cases (<1%) of diffuse large B-cell lymphoma (DLBCL), 9 cases (<1%) of Waldenstrom's macroglobulinemia (WM) and 3 cases (<1%) of B-cells prolymphocytic leukemia (B-PLL). 65 cases (4%) remained unclassified due to lack of histological and molecular data. 127 cases (7%) did not meet the diagnostic criteria of CLL established by Hallek et al but were classified as atypical CLL because of the detection of a clonal B-cell proliferation expressing CD5+ / CD23+ / CD43+ / CD10- / FMC7+ / CD79b+ with moderate or high intensity and light chain kappa or lambda with moderate or strong intensity (absence of molecular or histological argument of MZL or MCL was required). CD20 marker was highly expressed in 113 cases (89%) of atypical CLL. We particularly studied the 1532 cases (84%) of B-CLPD expressing CD5 (table). CD5+ CD23+ B-CLPD cases accounted for 1293 (84%) with 1153 cases of CLL, 13 cases of MCL and 127 cases of atypical CLL. CD5+ CD23- B-CLPD accounted for 239 cases (16%) with 72 cases of MCL, 158 cases of MZL, 4 cases of FL, 2 cases of WM, 2 cases of CD23- CLL and one case of B-PLL.CD5+ casesCD5+ CD23+ B-CLPDCD5+ CD23- B-CLPDTotalCLL115301153Atypical CLL1272129MCL137285MZL0158158FL044WM022B-PLL011Total12932391532 WHO classification of hematologic malignancies do not include atypical CLL as defined by a clonal proliferation of B-cells expressing CD5+, CD23+, cyclin D1- with no histological evidence of MZL or MCL, and which do not meet all the diagnostic criteria of CLL (Hallek et al, Blood 2008). This concept of atypical CLL, first described by Criel et al (BJH 1997), is particularly interesting, because such B-CLPD seems to have a different outcome as compared with CLL (Oscier et al, BJH 1997) and to have a different biological presentation with atypical morphology of CLL cells (Criel et al, BJH 1997), more frequent trisomy 12 (Matutes et al, BJH 1996) and a stronger intensity of CD20 (Ugo et al, Leuk Lymphoma 2006). Diagnosis of B-CLPD relies on a multidisciplinary approach combining morphological, immunophenotypic, molecular and histological analyses. Despite detailed information of these analyses, there are B-CLPD which remain unclassifiable according WHO classification, especially CD5 positive B-CLPD. The concept of atypical CLL seems to take all its meaning to help define such unclassifiable entities. Disclosures: No relevant conflicts of interest to declare.

Clinical characterization of first cycle reactions in patients with chronic lymphocytic leukemia treated with oblimersen

2006 ◽  
Vol 24 (18_suppl) ◽  
pp. 16514-16514
Author(s):  
K. S. Kolibaba ◽  
A. A. Chanan-Khan ◽  
G. Bociek ◽  
T. Boyd ◽  
J. Moore ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Ex Vivo ◽  
Phase 1 ◽  
Single Agent ◽  
Tumor Lysis Syndrome ◽  
Interleukin 2 ◽  
Serum Levels ◽  
Interferon Γ ◽  
Lymphocytic Leukemia ◽  
Bulky Disease

16514 Background: Serious adverse reactions during the first treatment cycle characterize many drugs used for chronic lymphocytic leukemia (CLL), including rituximab, alemtuzumab, flavopiridol, lenalidomide, and oblimersen. Termed cytokine release reactions (CRRs), these events have occurred more commonly in pts with bulky disease and elevated leukocyte counts, and some have been fatal. Rapid increases in serum levels of tumor necrosis factor-α, interleukin-6, interleukin-2, interferon-γ, and other cytokines may result in symptoms including fever, chills, nausea, vomiting, hypotension, and dyspnea. Ex vivo treatment of CLL cells with oblimersen induce release of vasoactive interleukin-8. First cycle reactions, including tumor lysis syndrome (TLS), proved dose-limiting in a phase 1–2 trial of oblimersen in pts with relapsed or refractory CLL. To characterize these reactions, we evaluated data from this single-agent study and from a phase 3 randomized clinical trial of oblimersen with fludarabine and cyclophosphamide (Flu/Cy). Methods: Data on investigator assessment of CRR and TLS were obtained from both studies, which included 155 pts; 40 were treated with oblimersen alone (3–7mg/kg/d by CIV for 5–7 days) and 115 with oblimersen (3 mg/kg/d × 7 days) in combination with Flu/Cy. Results: Of 145 pts treated with oblimersen 3mg/kg/d, either alone or with Flu/Cy, 2 pts (1.3%) experienced CRR and TLS, respectively. Two CRRs occurred in 10 pts treated with 4–7mg/kg/d. Reactions typically began 24–48 hours after starting treatment. CCR was usually associated with spiking fever, nausea, dehydration, rigors, and back pain. Rarely pts developed hypotension, acidosis, or reversible renal insufficiency, usually with a reduction in WBC. Symptoms lasted 1 day to several weeks (1 pt). In most cases, reactions were reversible with intensive supportive care, including IV fluids, pressors, and antibiotics; 1 death from TLS and 1 from CCR occurred during cycle 1. There was no definitive correlation with baseline measures of disease burden. Conclusion: First cycle oblimersen reactions are uncommon but represent a risk. Early symptoms (fever, nausea, and dehydration) require aggressive treatment, which may prevent progression of the reaction. [Table: see text]

scholarly journals Functional studies of the neoplastic B cells In chronic lymphocytic leukemia

2018 ◽  
Author(s):  
Ελισάβετ Χαρτοματσίδου
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Ex Vivo ◽  
Lymphocytic Leukemia ◽  
Wild Type ◽  
Functional Studies

1.H υπερέκφραση της μεθυλοτρανσφεράσης των ιστονών EZH2 στη Xρόνια Λεμφοκυτταρική λευχαιμία παρέχει προστασία από απόπτωση και συνδέεται με κλινική επιθετικότητα. Η μεθυλοτρανσφεράση των ιστονών ΕΖΗ2 είναιη καταλυτική υπομονάδα του κατασταλτικού συμπλόκου Polycomb 2 (PolycombRepressorComplex 2, PRC2) μέσω του οποίου καταλύει την τριμεθυλίωση της ιστόνης Η3 στο κατάλοιπο λυσίνη 27 (H3K27me3) επάγοντας αποσιώπηση γονιδίων. Η ΕΖΗ2 υπερεκφράζεται σε πλειάδα κακοηθειών, μεταξύ άλλων και κακοήθειες του λεμφικού ιστού και γενικά συσχετίζεται με δυσμενή πρόγνωση. Σε προκαταρκτική μελέτη μας δείξαμε για πρώτη φορά ότι η ΕΖΗ2 εκφράζεται στη χρόνια λεμφοκυτταρική λευχαιμία (ΧΛΛ), ιδίως σε περιπτώσεις που ανήκουν στο υποσύνολο #1, ομάδα ασθενών οι οποίοι χαρακτηρίζονται από δυσμενή κλινική πορεία. Στην παρούσα μελέτη επεκτείναμε την ανάλυση της έκφρασης της ΕΖΗ2 σε καλά χαρακτηρισμένη ομάδα 131 ασθενών με ΧΛΛ και διερευνήσαμε το λειτουργικό αντίκτυπό της στη συμπεριφορά των λευχαιμικών κλώνων. Η μελέτη περιλάμβανε: (i) ποσοτικοποίηση των mRNA μεταγράφων της ΕΖΗ2, (ii) προσδιορισμό της έκφρασης της ΕΖΗ2 σε επίπεδο πρωτεΐνης, (iii) μέτρηση επιπέδωντου δείκτη λειτουργίας της EZH2, H3K27me3, (iv) ανάλυση βιωσιμότητας ΧΛΛ κυττάρων με κυτταρομετρία ροής, (v) διαμόλυνση των ΧΛΛ κυττάρων με siRNA ειδικό για την EZH2 και τέλος, (vi) εφαρμογή φαρμακολογικών αναστολέων της ΕΖΗ2 σε κύτταρα ΧΛΛ. Τεκμηριώνουμε ότι η υπερέκφραση της EZH2 αποτελεί βασικό χαρακτηριστικό των κλινικά επιθετικών περιπτώσεων ΧΛΛ που φέρουν κυρίως αμετάλλακτα γονίδια IGHV, επηρεάζοντας τα επίπεδα της H3K27me3 στα λευχαιμικά κύτταρα και προσδίδοντάς τους έτσι πλεονέκτημα επιβίωσης και προστασία από απόπτωση. Στη συνέχεια, για να αποδείξουμε ότι η ΕΖΗ2 ευθύνεται για την αυξημένη βιωσιμότητα των κυττάρων, καταστείλαμε την έκφραση της ΕΖΗ2 με ειδικά siRNAs σε περιπτώσεις με ‘’υψηλή ΕΖΗ2’’και διαπιστώσαμε ότι μείωση της έκφρασης της ΕΖΗ2 επιφέρει επίσης μείωση των αντίστοιχων επιπέδων της H3K27me3, καθώς και επαγωγή απόπτωσης των κυττάρων της ΧΛΛ. Επιπλέον, δείξαμε ότι αυξημένη έκφραση της ΕΖΗ2 συσχετίζεται με ταχύτερη πρόοδο νόσου. Επίσης, παρουσιάζουμε προκλινικά δεδομένα που δείχνουν ότι η αναστολή της καταλυτικής ενεργότητας της ΕΖΗ2 με ειδικούς φαρμακολογικούς αναστολείς, όπως οι EPZ6438, GSK343 και GSK126, προάγει απόπτωση των κυττάρων της ΧΛΛ, καθώς και μείωση των επίπεδων της Η3Κ27me3, αναδεικνύοντας ένα νέο πιθανό θεραπευτικό στόχο για ασθενείς με επιθετική νόσο. 2. Συνεργική δράση των αναστολέων της άνοσης σηματοδότησης και της μεθυλοτρανσφεράσης των ιστονών EZH2 στη Xρόνια Λεμφοκυτταρική λευχαιμία: θεραπευτικές προεκτάσειςΗ ΕΖΗ2 υπερεκφράζεται σε κλινικά επιθετικές περιπτώσεις με ΧΛΛ και επάγει αυξημένη επιβίωση και προστασία από απόπτωση στα λευχαιμικά κύτταρα. Tα Β κύτταρα της ΧΛΛ λαμβάνουν σήματα επιβίωσης από το μικροπεριβάλλον μέσω πολλαπλών υποδοχέων, όπως ο Β κυτταρικός υποδοχέας (ΒΚΥ), οι υποδοχείς τύπου Toll (TLR) και ο CD40. Με βάση τα παραπάνω, στην παρούσα μελέτη εξετάσαμε εάν ερεθίσματα από το μικροπεριβάλλον μπορούν να επηρεάσουν την έκφραση της ΕΖΗ2 και κατ’ επέκταση τη βιωσιμότητα των κυττάρων της ΧΛΛ. Η ομάδα μελέτης καθορίστηκε με βάση την ικανότητα απάντησης στη διέγερση του ΒΚΥ: έτσι, επιλέχθηκαν για ανάλυση μόνο περιπτώσεις που απαντούσαν στην ενεργοποίηση του ΒΚΥ. Διαπιστώσαμε ότι ερεθίσματα του μικροπεριβάλλοντος μέσω του ΤLR9 και/ή CD40, αλλά ιδίως η διέγερση μέσω του υποδοχέα TLR9, οδηγεί σε αυξημένη έκφραση της ΕΖΗ2 και ενισχύει την επιβίωση του λευχαιμικού κλώνου. Σε αυτό το πλαίσιο, εξετάσαμε την επίδραση των αναστολέων της άνοσης σηματοδότησης στην έκφραση της ΕΖΗ2 στα ΧΛΛ κύτταρα καθώς και την πιθανή συνεργική δράση τους με αναστολείς της ΕΖΗ2. Αρχικά, μελετήσαμε εάν η θεραπεία με Ibrutinib μπορεί να επηρεάσει την έκφραση της EZH2 και τα επίπεδα της Η3K27me3 invivo. Η ομάδα μελέτης περιλάμβανε ασθενείς με ΧΛΛ οι οποίοι έλαβαν Ibrutinib ως μονοθεραπεία. Ανάλυση με ανοσοαποτύπωση western ανέδειξε μείωση των επιπέδων της πρωτεϊνικής έκφρασης της EZH2 καθώς και των επιπέδων της Η3Κ27me3 μετά από 1 μήνα θεραπείας συγκριτικά με το αντίστοιχο δείγμα πριν τη θεραπεία, ενισχύοντας τα προηγούμενα ευρήματά μας που συνδέουν τη σηματοδότηση των Β κυττάρων με την έκφραση και λειτουργία της ΕΖΗ2. Στη συνέχεια, ελέγξαμε με κυτταρομετρία ροής την επίδραση του Ibrutinib στη βιωσιμότητα των κυττάρων ex vivo σε περιπτώσεις ασθενών ΧΛΛ με υψηλά και χαμηλά επίπεδα EZH2 και διαπιστώσαμε ότι το Ibrutinib οδηγεί σε απόπτωση των ΧΛΛ κυττάρων μόνο στις περιπτώσεις με υψηλά επίπεδα ΕΖΗ2. Σε μια διαφορετική ομάδα ασθενών με ΧΛΛ οι οποίοι είχαν ελεγχθεί και βρεθεί να απαντούν σε διέγερση μέσω του TLR9 εξετάσαμε την πιθανή συνεργική δράση μεταξύ των αναστολέων της άνοσης σηματοδότησης Ibrutinib (IB) και Idelalisib (IDE) και των αναστολέων της EZH2 (GSK343, GSK126). Μετά από διέγερση με CpG (ειδικός προσδέτης για τον TLR9) επί 24 ώρες, ώστε να αυξηθεί η έκφραση της ΕΖΗ2, ακολούθησε μεμονωμένη επώαση με κάθε αναστολέα (IB, IDE, GSK343, GSK126) και συνδυαστική εφαρμογή των αναστολέων (IB/GSK343, IDE/GSK343, IB/GSK126). Μετά από 3 ημέρες καλλιέργειας αναλύθηκαν με κυτταρομετρία ροής τα επίπεδα βιωσιμότητας και Η3Κ27me3. Η μεμονωμένη αναστολή με IB, IDE, GSK343 και GSK126 οδήγησε σε απόπτωση των ΧΛΛ κυττάρων σε σχέση με ΧΛΛ κύτταρα χωρίς έκθεση σε αναστολείς (FD≥ 1.4), ενώ η συνδυαστική αναστολή με IB/GSK343, IDE/GSK343 και IB/GSK126 ήταν πολύ πιο αποτελεσματική, εφόσον οδήγησε σε ακόμα μεγαλύτερη μείωση της βιωσιμότητας και των επιπέδων της Η3Κ27me3 σε σύγκριση με τη μεμονωμένη αναστολή (FD≥1.5, p<0.05). Στη συνέχεια απομονώσαμε Β σπληνοκύτταρα από Tcl1 λευχαιμικά ποντίκια (το καλύτερα μελετημένο μοντέλο ποντικού στη ΧΛΛ) όπου παρατηρήσαμε αυξημένη έκφραση ΕΖΗ2 σε σχέση με κύτταρα από ποντίκια μάρτυρες (Wild Type, WT), καθώς επίσης αυξημένα επίπεδα πολλαπλασιασμού συγκριτικά με τα κύτταρα της ΧΛΛ. Τα προηγούμενα ευρήματα σχετικά με τη συνεργική δράση των αναστολέων άνοσης σηματοδότησης και της EZH2 επιβεβαιώθηκαν επίσης στα Β σπληνοκύτταρα από Tcl1 λευχαιμικά ποντίκια, με εμφανή μείωση επιπέδων πολλαπλασιασμού και βιωσιμότητας μετά από συνδυαστική δράση των αναστολέων συγκριτικά με τη μεμονωμένη επώαση. Τέλος, δείξαμε ότι η εφαρμογή φαρμακολογικών αναστολέων της EZH2 (GSK343 και GSK126) επάγει απόπτωση και μείωση στα επίπεδα H3K27me3 σε κύτταρα από περιπτώσεις ΧΛΛ που εμφάνισαν ανθεκτικότητα ή μειωμένη απόκριση στη θεραπεία με αναστολείς σηματοδότησης (IΒ ή IDE), παρέχοντας έτσι νέα δεδομένα για το σχεδιασμό και την παρακολούθηση των ασθενών με εξατομικευμένες θεραπευτικές στρατηγικές προσεγγίσεις.Συμπερασματικά, παρουσιάζουμε για πρώτη φορά ισχυρές ex vivo ενδείξεις ότι ο συνδυασμός φαρμακολογικών αναστολέων της άνοσης σηματοδότησης και αναστολέων της EZH2 έχει συνεργική δράση στη ΧΛΛ, υποδεικνύοντας μια εναλλακτική θεραπευτική στρατηγική για κλινικά επιθετικές περιπτώσεις ΧΛΛ.

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