Bortezomib Sensitizes Non-Hodgkin’s Lymphoma Cells to Apoptosis Induced by Antibodies to TRAIL Receptors R1 and R2.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4614-4614
Author(s):  
Mitchell Reed Smith ◽  
Fang Jin ◽  
Indira Joshi
Keyword(s):  
Growth Inhibition ◽  
Cell Lines ◽  
Annexin V ◽  
Hematologic Malignancies ◽  
Caspase Inhibitor ◽  
Apoptosis Induction ◽  
Minimal Growth ◽  
Trail Receptors ◽  
Independent Mechanism ◽  
Induction Of Apoptosis

Abstract TRAIL-R1 and -R2 signaling induces apoptosis via a pathway that activates caspase 8. The proteosome inhibitor bortezomib may act via several pathways. Agonistic antibodies to TRAIL-R1 and -R2 and bortezomib are in clinical trials in solid tumors and hematologic malignancies. To develop rational combinations for future clinical studies, we investigated the actions of these agents on non-Hodgkin s lymphoma (NHL) cell lines. The t(14;18)+, EBV- NHL cell lines DoHH2 and WSU-FSCCL were treated with agonistic monoclonal antibodies to TRAIL-R1 (HGS-ETR1) and -R2 (HGS-ETR2) (Human Genome Sciences, Rockville, MD) and/or bortezomib. While HGS-ETR 1 and HGS-ETR 2 are effective inducers of apoptosis in FSCCL, DoHH2, which expresses dim TRAIL-R1 (DR4, HGS-ETR1 target) and TRAIL-R2 (DR5, HGS-ETR2 target), shows minimal growth inhibition or apoptosis induction by HGS-ETR1 or HGS-ETR2. Bortezomib has modest effects on DoHH2 cells in growth inhibition and apoptosis assays. HGS-ETR1 and HGS-ETR2 induction of apoptosis in WSU-FSCCL is efficiently blocked by the caspase inhibitor ZVAD. In contrast, bortezomib effects are not blocked by ZVAD, indicating an independent mechanism of action. To determine if these separate pathways would provide enhanced combination activity, DoHH2 cells were pre-treated with bortezomib for 30 min, followed by incubation with HGS-ETR1 or HGS-ETR2. This led to supra-additive induction of apoptosis (annexin V staining). We conclude that bortezomib sensitizes DoHH2 cells to the action of HGS-ETR1 and HGS-ETR2. Further, bortezomib induces apoptosis in DoHH2 cells by an independent mechanism, and the combination of TRAIL-receptor signaling and bortezomib may be a useful combination to explore.

Simultaneous Targeting of Bcl-2/Bcl-Xl and MEK/ERK Results in Highly Synergistic Induction of Apoptosis in Human AML, Both in Vitro and In Vivo.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2757-2757
Author(s):  
Marina Konopleva ◽  
Michele Milella ◽  
Julie C Watts ◽  
Maria Rosaria Ricciardi ◽  
Borys Korchin ◽  
...  
Keyword(s):  
Growth Inhibition ◽  
Cell Line ◽  
Cell Lines ◽  
Single Agent ◽  
Mek Inhibitor ◽  
Human Leukemia ◽  
Apoptosis Induction ◽  
Mek Inhibitors ◽  
Synergistic Induction ◽  
Induction Of Apoptosis

Abstract Abstract 2757 Poster Board II-733 Previous experience from our group suggests that simultaneous inhibition of MEK-to-ERK signaling and interference with the anti-apoptotic activity of Bcl-2/Bcl-xL, using a variety of different agents, may result in synergistic anti-leukemic activity (Milella M, Blood 2002; Ricciardi MR, ASH 2004; Konopleva M, Cancer Cell 2006; Milella M, ASH 2008). Here, we screened AML cell lines (OCI-AML3, HL-60, MOLM-13, and U937) for the growth inhibitory/pro-apoptotic effects of the combination of the BH3 mimetic ABT-737 and the selective MEK inhibitor PD0325901. With the notable exception of U937 cells all the cell lines tested displayed highly synergistic growth inhibition/induction of apoptosis in response to the combination (CIs ranging from 0.01 to 0.43); the 25:1 and 1:2 ABT-737/PD0325901 ratios appeared to have optimal synergistic effects in OCI-AML3 and MOLM-13 cells, respectively. Cell growth inhibition was primarily due to the highly synergistic induction of apoptosis in sensitive cell line models. From a mechanistic standpoint, ABT-737 induced ERK activation and Mcl-1 protein expression, two putative resistance mechanisms, both of which were efficiently abrogated by co-treatment with PD0325901. In addition to the modulation of Mcl-1, both single agent PD0325901 and combined ABT-737/PD0325901 treatment rapidly (1-6 hrs) induced BimEL dephosphorylation and Bak expression, thereby contributing to Mcl-1 inactivation. To further assess the role of pro-apoptotic Bcl-2 family members in the observed proapoptotic synergism betweeen ABT-737 and PD0325901, we analyzed the effects of the combination in Bim-, Bak-, and Bax-KO MEFs, as well as in double (Bak/Bax) KO; while these experiments demonstrated that all three proapoptotic proteins play a role in apoptosis induction by combined ABT-737/PD0325901, siRNA-mediated silencing of either Bim or Bak clearly indicated Bim as the most important player in the AML cell line OCI-AML3. In addition to cell line models of leukemia, striking apoptosis induction (20-75% net apoptosis induction) was also observed with the combination of Bcl-2 and MEK inhibitors in ex vivo-cultured primary AML samples (n=8); most interestingly, the ABT-737/PD0325901 combination appeared to selectively kill leukemic stem cells, with < 20% of CD34+/CD38- cells surviving after exposure to relatively low doses of the combination (50 nM for each agent). Finally, we tested the combination of ABT-737 and the MEK inhibitor CI-1040 in nude mice injected with GFP/luciferase bearing MOLM-13 human leukemia cells. Two weeks after leukemia transplantation, mice were randomized and treated with liposomal ABT-737 (i.v. 20 mg/kg, qod for three weeks), CI1040 (i.p. 50 mg/kg qod for three weeks), ABT-737 in combination with CI1040 (ABT-737 + CI1040), or with empty liposomes (i.v.; control). Engraftment of MOLM-13 cells was shown by immunohistochemical detection of GFP-positive cells in the spleen of control mice five weeks after transplantation. Notably, while control and CI1040 treated mice demonstrated progressive increases in leukemia-derived bioluminescence, ABT-737 treated mice, and to a greater extent ABT-737 + CI1040 treated mice, appeared to resist tumor burden progression. In addition, quantitation of leukemia-derived bioluminescence demonstrated that ABT-737 + CI1040 treated mice had significantly (p<0.00001) lower leukemia burden than control mice or ABT-737 treated mice at all time points (7, 14 and 21 days of treatment). Overall our data demonstrate that an anti-apoptotic crosstalk between the Bcl-2 and the MEK/ERK pathway is operative in AML cells and could be exploited therapeutically by targeting both pathways simultaneously. The combination of BH3 mimetics (such as ABT-737) and MEK inhibitors warrants clinical testing as a novel therapeutic strategy for patients with AML. Disclosures: No relevant conflicts of interest to declare.

Pre Clinical mTOR-Inhibition of Acute Lymphoblastic Leukemia Cells Synergizes with Pro-Apoptotic Target Therapy Through Mcl-1 Down-Regulation,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3581-3581
Author(s):  
Stefano Iacovelli ◽  
Maria Rosaria Ricciardi ◽  
Paola Bergamo ◽  
Cinzia Rinaldo ◽  
Roberto Licchetta ◽  
...  
Keyword(s):  
Growth Inhibition ◽  
Cell Lines ◽  
Prolonged Exposure ◽  
Jurkat Cells ◽  
Combined Effects ◽  
Apoptosis Induction ◽  
Down Regulation ◽  
Mtor Inhibition ◽  
Resistant Phenotype ◽  
Induction Of Apoptosis

Abstract Abstract 3581 ALL cells are frequently characterized by deregulation of several intracellular signaling pathways involved in the control of proliferation and apoptosis. Among these, the constitutive activation of the mTOR signaling cascades has been frequently reported in the majority of ALL patients and molecular therapies targeting mTOR have been therefore proposed with variable preclinical efficacy, generally inhibiting proliferation in a large number of cell lines and only in some instances leading to apoptosis. To improve pro-apoptotic effects of the mTOR inhibition, especially in ALL cells, a combined Bcl-2 family inhibition approach can be explored, based on the pivotal role of Bcl-2 in lymphoproliferative disease. In addition, Mcl-1 protein levels may be controlled by the AKT/mTOR pathway, since the prosurvival AKT, negatively regulate Gsk-3. In fact, following AKT inactivation, Gsk-3 promotes apoptosis by Mcl-1 phosphorylation and its proteasome degradation. Moreover, Mcl-1 overexpression is a know resistance mechanism to the BH-3 mimetic, the ABT-737. Interestingly prolonged exposure to mTOR inhibitor rapamycin is reported to inactivate the AKT signaling pathway. Aim of this study was to evaluate the combined effects of mTOR (CCI-779) and Bcl-2/Bcl-XL (ABT-737) inhibition in ALL cell lines and primary samples (21). In MOLT-4 cell line exposure to CCI-779, up to 72 hours, induced a flat dose-response curve (35–55% growth inhibition) at concentrations ranging between 1 and 5000 nM and apoptosis induction was not seen until 5000 nM. In CEM-S, CEM-R and JURKAT cell lines, only minor cytostatic effects were observed until 20000 nM. In MOLT-4 cells, ABT-737 induced dose and time-dependent growth inhibition (IC-50= 198nM) followed at higher concentrations (250–500nM) by induction of apoptosis. In contrast, the CEM-S, CEM-R and JURKAT cells, proved resistant (IC-50 >5 μM), displaying Mcl-1 overexpression. We therefore investigated in these resistant cell lines the combined effects of CCI-779+ABT-737. The JURKAT cells showed a significantly higher (p< 0.01) induction of apoptosis following exposure to ABT-737 and CCI-779 (both at 1000nM), as compared to single agents. Similar effects were observed in CEM-R cells exposed to the drug combination (CCI-779 5000nM and ABT-737 1000nM) (p<0.01). In both cell lines, CCI-779 exposure was correlated with down-regulation of the Mcl-1 protein, mediated by proteasome degradation. This effect was associated with AKTS473 de-phosphorylation, due to CCI-779 prolonged exposure. The observed Mcl-1 decrease was, however, not seen in the resistant phenotype CEM-S cell line. Further RNA interference of Mcl-1 did not convert the resistant phenotype. Primary ALL cells exposed to CCI-779 (5000 nM), showed in a minority (4/21) of samples only a weak apoptosis induction (sub-G1 peak< 20%), while a stronger effect (> 40%) was observed in 15/21 treated with ABT-737 (50nM) (Fig.1). The remaining 6/21 samples, proved resistant to ABT-737; in two of these, interestingly, the combined treatment overcame resistance, inducing Mcl-1 down-regulation and apoptosis induction. In summary, the Bcl-2/Bcl-XL (ABT-737) inhibition is an active proapoptotic treatment of ALL, in addition the combined use of mTOR (CCI-779) inhibition may revert ABT-737 resistant phenotypes in a proportion of ALL, via AKT inactivation and Mcl-1 degradation. However, different resistant mechanisms involved in ALL cells (CEM S), prompt further investigation. Disclosures: Foà: Bristol-Myers Squibb: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Preclinical Study to Sensitize Acute Lymphoblastic Leukemia Primary Cells by Combined mTOR and BCL-2 Inhibition with CCI-779 and ABT-737.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 985-985
Author(s):  
Maria Rosaria Ricciardi ◽  
Samantha Decandia ◽  
Paola Bergamo ◽  
Cinzia De Benedictis ◽  
Antonella Vitale ◽  
...  
Keyword(s):  
Growth Inhibition ◽  
Lymphoblastic Leukemia ◽  
Philadelphia Chromosome ◽  
Annexin V ◽  
Fold Increase ◽  
Jurkat Cells ◽  
Apoptosis Induction ◽  
Cell Growth Inhibition ◽  
Resistant Phenotype ◽  
Induction Of Apoptosis

Abstract Abstract 985 Poster Board I-7 Acute lymphoblastic leukemia (ALL) is characterized by a high relapse rate, especially in adult patients, with the majority developing eventually chemoresistance. Therapeutic strategies based on targeted therapy have improved survival especially in Philadelphia-chromosome positive patients. We have previously demonstrated that ABT-737 (kindly provided by Abbott Laboratories), a Bcl-2/Bcl-xL (BH3 mimetic) inhibitor, exerts potent cell growth inhibition and apoptosis induction in ALL cell lines and primary samples (Blood, 2007; 110:53a). However, a resistant phenotype, mostly characterized by Mcl-1 overexpression, has been identified for this drug. In this study we have explored the possibility of extending our previous observations by modulating in addition to apoptosis, also aberrant proliferative signals. Since Temsirolimus (CCI-779), a PI3/AKT/mTOR inhibitor, has been reported to induce growth arrest and apoptosis in preclinical models of ALL, we have investigated in ALL cell lines and primary human ALL cells the effect on cell proliferation and apoptosis of CCI-779 alone and in combination with ABT-737. Exploring CCI-779 activity in ALL cells as a single agent, a biphasic dose response was observed in MOLT-4 cells (IC50: 9865 nM), with a flat curve at concentrations (35-55% of inhibition) ranging between 1 nM and 5000 nM, and a more pronounced growth inhibition at concentrations ≥10000 nM, as demonstrated by the MTT based-assay. At these concentrations, induction of apoptosis was not seen up to 5000 nM, while at 24h the S-phase decreased from 37.6% ± 3.2 (vehicle) to 26.1% ±7.8 (p=0.04). Conversely, ABT-737 induced dose- and time-dependent growth inhibition in MOLT-4 cells (IC-50=198nM). Apoptosis induction, as measured by Annexin-V positivity, was not seen at lower concentrations (50 nM), but required higher concentrations of ABT-737 (controls from 11.9% ± 2, ABT-737 at 500 nM 62.7% ± 18.2). Using concentrations that only induce minimal apoptosis (1.3 and 1.4 fold increase with CCI-779 at 5000 nM and 50 nM of ABT-737, respectively), the combined treatment induced a 3.2 fold increase (p= 0.05) after 48 hours. These synergistic effects on induction of apoptosis were not seen on cell cycle modulation. Exploring then the CCI-779 activity on the T-ALL Jurkat cells, a scalar effect on cell growth inhibition was seen at increasing concentrations (from a 34%, to 46% and to 81% at 10, 1.000 and 15.000 nM, respectively). Jurkat cells were resistant to ABT-737 (IC-50=66 uM). This resistant phenotype was however converted to sensitivity by the combined use of ABT-737 and CCI-779 (1000 nM) which increased Annexin-V positive cells from 6.3% ± 1.1 (vehicle) to 10.5 ± 1.5 (CCI-779), 23.7 ± 1.5 (ABT-737) and 54 ± 5.9 (CCI-779 + ABT-737) (p=0.04). Thus, a synergist effect on apoptosis was found in T-ALL by combining CCI-779 and ABT-737. We also confirmed in this resistance model the lack of enhanced cell cycle effects. We then evaluated primary ALL cells obtained from six Philadelphia-chromosome negative patients with B-lineage ALL (5) and T-ALL (1) by treating enriched lymphoblasts with CCI-779 (ranging from 5.000 to 10.000 nM) and ABT-737 (ranging from 50 to 100 nM) alone and in combination. Although ABT-737 was generally effective in inducing apoptosis, an increase above 75% of the sub-G1 peak was seen in 2/6 and in 3/6 samples exposed to CCI-779 and to ABT-737, respectively, while the combination of both inhibitors was effective in 4/6 primary cases. In fact, the combination with CCI-779 increased apoptosis from 14.5% (vehicle), to 28.6% (50 nM of ABT-737), 33.3% (5000 nM of CCI-779), 71.8% (ABT-737 + CCI-779). In conclusion, we demonstrated that CCI-779 can potentiate the effect of ABT-737 in ALL cells. Disclosures: Petrucci: Celgene: Honoraria; Janssen Cilag: Honoraria.

scholarly journals 2193

2017 ◽  
Vol 1 (S1) ◽  
pp. 58-59
Author(s):  
Houda Alachkar ◽  
Martin Mutonga ◽  
Amanda de Albuquerque ◽  
Rucha Deo ◽  
Gregory Malnassy ◽  
...  
Keyword(s):  
Cell Viability ◽  
Cell Lines ◽  
Therapeutic Target ◽  
Leucine Zipper ◽  
Lymphoblastic Leukemia ◽  
Annexin V ◽  
Hematologic Malignancies ◽  
Cytotoxic Activities ◽  
Knock Down ◽  
Protein Levels

OBJECTIVES/SPECIFIC AIMS: Unlike the high cure rates (90%) of children with acute lymphoblastic leukemia (ALL), that of adults is still lagging behind and better therapies are needed. Maternal embryonic leucine-zipper kinase (MELK) is aberrantly upregulated in cancer, and implicated in cancer stem cell survival. A recent study has identified FOXM1, a MELK substrate, as a therapeutic target in B cell ALL (B-ALL). Thus, we hypothesized that MELK may act as a therapeutic target in ALL via targeting FOXM1 activity. METHODS/STUDY POPULATION: Western blot and qPCR were used to assess MELK expression in 14 ALL cell lines. Knock-down and kinase inhibition approaches targeting MELK expression and function, followed by CCK-8 and Annexin V (flow cytometry) assays to measure cell viability and apoptosis, respectively. RESULTS/ANTICIPATED RESULTS: MELK was significantly upregulated in patients with ALL (oncomine data analysis). MELK was also significantly higher in B-ALL and T-ALL cell lines compared with that in blood cells of healthy donors. MELK knock-down significantly decreased cell viability (40%–70%, p<0.05, Fig. 1) in ALL cells, and induced apoptosis (~40%). OTS167, a potent MELK inhibitor exhibited cytotoxic activities in both B and T-ALL cells. The IC50 of OTS167 ranged from 20 to 60 nM; we also found a significant increase in apoptosis (p<0.05). Mechanistically, MELK inhibition resulted in decrease of FOXM1 protein levels 3 hours post-treatment. DISCUSSION/SIGNIFICANCE OF IMPACT: MELK is highly expressed in ALL and represents a novel therapeutic target likely via modulating FOXM1 activity. Functional and mechanistic studies will complement and ensure the success of the undergoing Phase I/II clinical trial of OTS167 in patients with refractory or relapsed AML, ALL, and other advanced hematologic malignancies.

Targeting xIAP Is More Important Than Bcl-Xl in Lymphomas with Constitutive Activation of NF-kB.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2390-2390
Author(s):  
Yanjuan He ◽  
Joan Cain ◽  
Lee Ratner ◽  
Leon Bernal-Mizrachi
Keyword(s):  
Cell Lines ◽  
Annexin V ◽  
Fold Increase ◽  
Daudi Cell ◽  
Xiap Expression ◽  
Apoptotic Effect ◽  
Effective Doses ◽  
Induction Of Apoptosis ◽  
Apoptotic Pathways

Abstract Pathways resulting in resistance to apoptosis are essential to the process of lymphomagenesis. One such pathway, the nuclear factor-kB (NFkB), has been shown to be a key element in coordinating the anti-apoptotic effect of these malignancies. However the mechanisms used by which NFkB prevents apoptosis are not well understood. It has been suggested that NFkB inhibits activation of the intrinsic, extrinsic and common apoptotic pathways. Previous work in our lab using two different virally mediated lymphoma models (Tax/HTLV1 and LMP1/EBV driven tumors) has identified two candidates that could explain these results: X chromosome-linked inhibitor of apoptosis (xIAP) and BCL-xL. Although the current literature extensively demonstrates the role of BCL-xL in lymphomas, little is known about the importance of xIAP in these malignancies. To answer this question we tested the apoptotic effect of etoposide or tumor necrosis factor (TNF) after knocking down bcl-xL and xIAP expression in our lymphoma models (SC and Daudi cell lines) using a lentivirus expressing siRNAs. After 24 hours of treatment with etoposide and TNF, we measured apoptosis by flow cytometry using double staining with Annexin V-Alexa Fluorescense and propidium iodide. Interestingly, xIAP siRNA-expressing cell lines demonstrated 2–4 fold increase in the induction of apoptosis after treatment with etoposide as compared to a nearly 2 fold increase in those expressing Bcl-xL siRNA (see Table below). No synergism was seen after treatment with TNF. Based on this finding, we then tested a novel small molecule, homolog smac, (SHC, kindly provided by Dr. PG Harren) to determine the possible therapeutic effect of xIAP inhibitors. After titration, the two most effective doses were selected (25 μM and 50 μM) to treat Daudi cell lines for 24hrs, with either etoposide or TNF. At doses of 25 μM , we observed a 2 fold increase in the induction of apoptosis produced by etoposide compared to that seen in control (DMSO + etoposide) or SHC alone and no synergism with TNF confirming the siRNA data. More importantly, at doses of 50 μM, SHC alone demonstrated activity with a 5 fold increase in apoptosis and a nearly 10 fold increase as compared to control (DMSO) when etoposide was added. Overall, we have demonstrated that xIAP and bcl-xL are important in mediating NFkB-resistance to apoptosis. However, our findings suggested that xIAP is a more potent anti-apoptotic signal and opens the door for further drug development aimed at testing xIAP-inhibitors in lymphomas. Induction of Apoptosis in xIAP or Bcl-xL siRNA expressing cell lines siRNA/Compound Etoposide TNF Untreated xIAP 43.1 ± 17.6 17.04 ± 1.4 14.3 ± 2 SC Bcl-xL 18.39± 3.7 9.4 ± 0.22 12.5 ± 2.7 Luc/DMSO 14.9 ± 1.8 14.4 ± 5.6 14.03 ± 1.25 xIAP 9.2 ± 3.2 4.7 ± 0.48 4.6 ± 0.44 Bcl-xL 8.9 ± 0.5 5.3 ± 1.7 4.16 ± 0.4 Daudi Luc/DMSO 5.49 ± 1.71 4.28 ± 0.5 6.2 ± 0.9 SHC 25 μM 20.07 ± 4.8 12.8 ± 3.9 12.1 ± 3.2 SHC 50 μM 47.7 ± 14.55 38.3 ± 0.99 32.7 ± 8.99

Dynamic Assessment of Apoptosis for In Vitro Design of Bortezomib Combination Therapies for Lymphoid Malignancies.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2494-2494
Author(s):  
Lauren C. Wallis ◽  
Matthew J. Streetly ◽  
Rebecca Auer ◽  
John Gribben ◽  
Dean Zhang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Real Time ◽  
Flow Cytometric Analysis ◽  
Annexin V ◽  
Growth Characteristics ◽  
Cytotoxic Agents ◽  
Alamar Blue ◽  
Apoptosis Induction ◽  
Flow Cytometric ◽  
Induction Of Apoptosis

Abstract Conventional techniques for assessing drug response and apoptosis induction rely on static assessment of cellular changes at predetermined time points (e.g. detection of exposed membrane phospholipids by Annexin V). The Kinetics of Optical Response assay (KOR) is a new technique that detects induction of apoptosis dynamically. It employs a spectrophotometric methodology to detect changes in optical density associated with membrane blebbing related to growth and death, allowing detection of apoptosis in real time. The KOR assay has already predicted the response to cytotoxic agents of AML cell lines and primary samples. This study uses the KOR assay in lymphoid malignancy and shows sensitivity to apoptosis induction by conventional and novel agents including bortezomib. The lymphoma cell line DOHH2 (t(14;18)), U266 (myeloma), K562 (CML) and primary CLL cells were used in this study with HL60 (AML) as a control. Cells were seeded in 96 well plates and treated with a variety of drugs alone or in combination (cytarabine, fludarabine, doxorubicin, daunorubicin, etoposide, melphalan, bortezomib) at multiple concentrations. Measurements were made at 5 min. intervals for up to 48 hrs and analysed using KORSoft™ software to generate apoptotic response curves. To validate this approach conventional techniques were used for comparison (Alamar Blue for cytotoxicity and flow cytometric analysis of cell cycle and apoptosis using propidium iodide and Annexin V staining respectively). The KOR assay can show changes in growth characteristics, induction of apoptosis and necrosis in response to drugs permitting a continuous analysis for maximum sensitivity (Smax). DOHH2 was found to be dose responsive to four of the drugs used, with the Smax for 10μM daunorubicin at 6 hours (48%), 1μM doxorubicin at 8 hours (38%), 100μM etoposide at 8 hours (52%), and minimally to 100μM cytarabine at 16 hours (21%). There was no effect from fludarabine. The addition of bortezomib increased Smax to 89% with etoposide and to a lesser degree with the other cytotoxic drugs. U266 showed a similar spectrum of results with greatest Smax with 100μM melphalan at 9 hours (57%) enhanced to 78% with the addition of bortezomib. There was minimal response to cytarabine and fludarabine. Parallel flow cytometric analysis using Annexin V and PI showed similar results to those from the KOR assay confirming the assessment of apoptosis to be valid. Cell cycle analysis showed an increased sub-G1 peak in keeping with apoptosis at times of Smax assessed by the KOR assay. The Alamar Blue cytotoxicity assay showed a dose dependent decrease in cell proliferation in response to increasing drug dose again paralleling other apoptosis measurements implying an apoptotic effect due to drug action and correlate well with those from the KOR assay. Primary CLL samples following CD19 selection were cultured with and without IL4 and exposed to the KOR assay with cytotoxics and bortezomib. Culture with IL4 alone gave good growth characteristics and revealed the combination of etoposide and bortezomib to provide the best induction of apoptosis (Smax 82%) compared to etoposide (26%) or bortezomib (32%) alone. The KOR assay is a microtitre approach to the assessment in real time of apoptosis. This study suggests the combination of bortezomib and etoposide is effective for lymphoma. Such approaches can accelerate the development of effective clinical trials.

Roscovitine Sensitizes Leukemia and Lymphoma Cells to TRAIL-Induced Apoptosis and Enhances Cell-Mediated Cytotoxicity

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 596-596
Author(s):  
Jan Molinsky ◽  
Marie Markova ◽  
Magdalena Klanova ◽  
Michal Koc ◽  
Lenka Beranova ◽  
...  
Keyword(s):  
Clinical Trials ◽  
Cell Lines ◽  
Low Dose ◽  
Hematologic Malignancies ◽  
Additive Effect ◽  
Cytotoxic Agents ◽  
Potential Contribution ◽  
Primary Cells ◽  
Trail Receptors ◽  
Induced Apoptosis

Abstract Abstract 596 Roscovitine is a selective inhibitor of cyclin-dependent kinases (CDK) and it is under evaluation in several clinical trials in the treatment of diverse cancers. TNF-related apoptosis inducing ligand (TRAIL) is a death ligand important for tumor immunosurveillance with selective antitumor activity and minimal toxicity toward tissues. Soluble TRAIL is also under evaluation in several clinical trials. Unfortunatelly, many cancers are resistant to TRAIL. To circumvent TRAIL resistance, there is effort to combinate TRAIL with other cytotoxic agents. By measuring apoptosis and proliferation, we demonstrated that combination of low dose roscovitine and low dose TRAIL (low dose= up to 30% of apoptotic cells after 24h treatment) is synergistic in 20 of 21 tested hematologic cell lines including TRAIL resistant cell lines. Moreover, this combination was tested on primary cells from 9 patients with hematologic malignancies with synergism in 4 of 8 samples from patients with acute myeloid leukemia (AML) and 1 sample from patient with mantle cell lymphoma. Remaining 4 AML samples showed additive effect. Based on these results, we decided to explore molecular mechanisms responsible for the synergism between roscovitine and TRAIL using TRAIL-resistant K562 cells. Despite decreased mRNA, the surface expression of TRAIL receptors remained unaffected after 24h roscovitine treatment. Immunoprecipitation of death-inducing signaling complex (DISC) revealed distinct proapoptotic changes (enhanced CASP8 and 10, reduced FLIP at 12 and 24h). These proapoptotic changes suggested that roscovitine might synergize with other death ligands acting through the DISC, namely TNF and FASLG. Indeed, roscovitine significantly sensitized diverse cell lines (K562, DOHH2, RAMOS) to TNF or FASLG-induced apoptosis. We subsequently proved that pretreatment of the cells (K562, DOHH2, RAMOS) with roscovitine increased by approx. 20% the level of cell-mediated cytotoxicity (peripheral blood mononuclear cells from a healthy volunteer marked with carboxyfluorescein succinimidyl ester). Thus, proapoptotic changes of the DISC seem to play essential role in mediating roscovitine-induced sensitization to TRAIL. Despite detected alterations of the DISC, we decided to unveil additional potential changes in the protein levels of key apoptotic regulators by western blotting at 1.5, 3, 6, 12 and 24h timepoints. Like Ortiz-Ferron et al. we detected gradual downregulation of MCL1 that peaked at 12h, followed, however, by substantial upregulation at 24h. We proved that even at this point, i.e. at 24h exposure to roscovitine, the cells were sensitized to TRAIL-induced apoptosis. The role of MCL1 in mediating the proapoptotic change thus remains elusive. BCL-XL showed similar kinetics as MCL1. Several proapoptotic proteins were overexpressed (BAK and BAD at 1.5h, and PUMA at 1.5h and 24h). Gene-expression profiling unveiled additional changes that might contribute to sensitization to TRAIL, e.g. upregulation of proapoptotic death inducer-obliterator 1 (DIDO1) and downregulation of antiapoptotic DNA-damage-inducible transcript 4 (DDIT4). In contrast to TRAIL (and the other death ligands) roscovitine showed only additive effect or even antagonism with the tested genotoxic agents (cytarabine, doxorubicin, fludarabine, etoposide, cisplatin) probably due to the inhibition of CDK2 by roscovitine (Yu et al., Yanjun et al.). We demonstrated that combination of roscovitine and TRAIL is synergistic in hematologic cell lines and primary cells. In addition, roscovitine was shown to have potent immunostimulatory effect by increasing cell-mediated cytotoxicity. Based on our results we suggest that roscovitine-induced sensitization to TRAIL-triggered apoptosis was mediated by proapoptotic changes of the DISC with potential contribution of the proapoptotic changes in the protein expression of the apoptotic regulators (MCL1, BCL-XL, PUMA, BAK, BAD). We also suggest that roscovitine-induced increase in cell-mediated cytotoxicity, known to be mediated in part through death ligands, was also a consequence of the proapoptotic alteration of the DISC. Roscovitine, as a single agent, or in combination with TRAIL, might have a role in the experimental treatment of selected hematologic malignancies. Financial Support: LC 06044, MSM 0021620806, MSM 0021620808, GAUK 259211/110709, SVV-2010-254260507, IGA MZ NS/10287-3 Disclosures: No relevant conflicts of interest to declare.

CRM1 Is Highly Expressed in Myeloma Plasma Cells and Its Inhibition by KPT-SINE Induces Cytotoxicity by Increasing p53 in the Nucleus of Multiple Myeloma (MM) Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1852-1852 ◽  
Author(s):  
Malathi Kandarpa ◽  
Stephanie J Kraftson ◽  
Sean P Maxwell ◽  
Dilara McCauley ◽  
Sharon Shacham ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Cell Lines ◽  
Tumor Suppressors ◽  
Nuclear Export ◽  
Plasma Cells ◽  
Annexin V ◽  
Apoptosis Induction ◽  
Advisory Committees ◽  
Tumor Suppressor Proteins ◽  
Rpmi 8226

Abstract Abstract 1852 Background: CRM1 (XPO1, exportin) is a nuclear export protein which controls the nuclear-cytoplasmic localization of multiple tumor suppressor proteins and cell proliferation pathways including p53, p21, PI3K/AKT/FOXO, Wnt/ß-catenin/APC, topoisomerase II, and NF-κB/I-κB. Transport of nuclear proteins to the cytoplasm can render them ineffective as tumor suppressors or as targets for chemotherapy. Small molecule, selective inhibitors of nuclear export (SINE) that block CRM1-dependent nuclear export can force the nuclear retention of tumor suppressor proteins, thus rendering cancer cells more susceptible to apoptosis and responsive to other chemotherapy. In this study we evaluated CRM1 as a potential target in MM and the effect of SINE on the activity of established anti-myeloma agents currently in use in treatment of MM. KPT-276 is the lead CRM1 inhibitor being investigated which will be submitted for IND in 2012. Methods: To evaluate expression of CRM1, bone marrow aspirates from MM patients and tonsil tissue from normal patients were enriched for plasma cells (PC) and proteins from cell lysates were separated by SDS-PAGE followed by immunoblotting with CRM1 antibodies. In functional experiments, isolated fresh MM PCs from patients, and NCI-H929, MM1.S, MM1.R and RPMI-8226 cell lines were cultured in RPMI-1640 with 10–15% serum. Cells were treated for 24–72 hrs with CRM1 inhibitors KPT-SINE compounds with or without bortezomib and dexamethasone and were analyzed for cytotoxicity by MTT assay. Drug concentrations for combination experiments were chosen to be at or below IC50 for each individual drug. Apoptosis induction in primary MM cells and cell lines was studied by Annexin V labeling and flow cytometry. Cell lysates from primary MM PCs and cell lines were prepared after treatment with KPT-SINE and were used to determine the expression of p53 and CRM1. Results: Primary MM plasma cells derived from naïve, previously untreated patients show 4–20 fold higher CRM1 protein expression, compared to normal peripheral blood mononuclear cells (PBMCs) and normal tonsilar PCs. Dose response analysis of KPT-SINE compounds in myeloma cell lines showed potent activity with IC50s in the range of 10–100nM. The lead compound KPT-276 had an IC50 of <100 nM in NCI-H929, MM1.S, MM1.R and RPMI-8226 cells. Functional studies in MM patient plasma cells showed that in vitro inhibition of CRM1 with related SINEs KPT-185, −225 or −276 increase apoptosis induction as measured by Annexin V assay. In addition, the inhibition of CRM1 with KPT-SINE results in a dose-dependent increase in levels of nuclear as well as total p53 in MM patient plasma cells within 48 hrs. When combined with proteasome inhibitors like bortezomib and/or dexamethasone, KPT-SINE compounds potently increase the cellular cytotoxicity of these drugs in MM cell lines. Mechanism of activity of drug combinations is under investigation in MM cell lines and MM patient plasma cells. Conclusions: MM plasma cells express CRM1 that is functionally active and therefore is a valid target in the treatment of myeloma. Moreover, higher expression of CRM1 in malignant plasma cells compared to normal PBMCs and normal PCs suggests possibility of therapeutic index. Early mechanistic studies indicate that CRM1 inhibition can lead to an increased expression of p53 (and other tumor suppressors) and its nuclear localization in myeloma cells and therefore might serve as a mechanism for the activity of CRM1 inhibitors in MM. Potentiation of cytotoxicity of bortezomib and dexamethasone by KPT-SINE suggests that these drugs might be useful in treating MM refractory to currently used agents and provide rationale for combining inhibitors of nuclear transport with other drugs. Disclosures: Off Label Use: KPT-SINE family of drugs are not approved for the treatment of multiple myeloma. These drugs have a novel mechanism and are in pre-clinical development for the treatment of several malignancies. McCauley:Karyopharm Therapeutic Inc.: Employment. Shacham:Karyopharm Therapeutics Inc.: Employment. Kauffman:Karyopharm Therapeutics Inc.: Employment. Jakubowiak:Exelixis: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Onyx Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Millennium Pharmaceuticals, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Ortho Biotech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

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