The Molecular Basis for BCL-2 Oncogene Addiction in CLL.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5008-5008
Author(s):  
Anthony Letai ◽  
Michael Certo ◽  
Jennifer R. Brown ◽  
Victoria Moore
Keyword(s):  
Cytochrome C ◽  
Cell Lines ◽  
Protein Function ◽  
Ex Vivo ◽  
Single Agent ◽  
Myeloma Cell ◽  
Cytochrome C Release ◽  
Bh3 Domain ◽  
Apoptotic Protein

Abstract CLL cells consistently express BCL-2 at a high level. Using a compound demonstrated to antagonize BCL-2 function, ABT-737, we show that CLL cells in short-term primary culture are uniformly sensitive to single-agent BCL-2 antagonism, with EC50’s in the 10 nM range. To understand the mechanism of CLL sensitivity to this compound, we studied mitochondria from these primary CLL cells. We employed a panel of peptides derived from the BH3 domain of pro-death BH3-only proteins, certain of which selectively inhibit BCL-2 function in vitro. We demonstrate that those with activity against BCL-2 in vitro induce cytochrome c release, a hallmark of the mitochondrial dysfunction during apoptosis. A non-malignant cellular model of BCL-2 dependence revealed that binding of the pro-apoptotic protein BIM to BCL-2 correlated with sensitivity to BCL-2 inhibitors. We likewise discovered that BCL-2 binds and sequesters BIM in CLL cells. In contrast to the BCL-2 dependent CLL cells, we examined myeloma cell lines with defined MCL-1 dependence and relative BCL-2 independence. These cell lines were 100 fold less sensitive to ABT-737. Furthermore, in mitochondrial assays only BH3 peptides which interact with MCL-1 induce cytochrome c release. These experiments reveal that cellular requirement for an anti-apoptotic protein function can be deduced from a functional mitochondrial requirement determined using our panel of BH3 peptides. In CLL, and perhaps other malignancies, it appears that BCL-2 maintains survival by sequestering pro-apoptotic proteins like BIM. Inhibition of this sequestration provokes cell death ex vivo, even with a single agent BCL-2 inhibitor. Reliance on BCL-2 for survival may be an Achilles heel for CLL and other cancers.

Characterization of Bortezomib Resistance in Multiple Myeloma Cell Lines

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2639-2639
Author(s):  
Silvia CW Ling ◽  
Angela M Nikolic ◽  
Ammira Al-Shabeeb ◽  
Edwin Lau ◽  
Phoebe Joy Ho ◽  
...  
Keyword(s):  
Cell Lines ◽  
De Novo ◽  
Single Agent ◽  
Active Form ◽  
Myeloma Cell ◽  
Cross Resistance ◽  
Parent Cell ◽  
Mrna Levels ◽  
Adherent Growth

Abstract Bortezomib is the first proteasome inhibitor in clinical use. It is particularly effective in myeloma compared with other cancers; as a single agent, its response rate in relapsed myeloma is about 40%. Nevertheless, a large proportion of patients are primarily resistant and acquired bortezomib resistance is also an emerging issue. The mechanism of bortezomib resistance in myeloma remains elusive. We have previously shown that bortezomib sensitivity correlates inversely with the levels of XBP-1 mRNA in vitro and in myeloma patients. XBP-1 is a major regulator of the unfolded protein response (UPR), which is essential for immunoglobulin folding and assembly, and plasma cell development. Previous studies suggested that immunoglobulin production sensitises myeloma cell lines to proteasome inhibitors (S Meister, et al., 2007, Cancer Res 67:1783). We hypothesize that dependence on XBP-1 and the UPR mediate sensitivity to bortezomib. The goal was to develop bortezomib resistant myeloma cell lines, study their phenotype, characterise the mechanism of resistance and, in particular, study the changes in the UPR. Bortezomib resistant sublines (9 in total) were derived from 4 myeloma cell lines (KMS-11, H929, U266 and OPM2), by long term exposure to bortezomib. The fold resistance varies from 3 to 12 and is stable for at least 4 passages without bortezomib. During the evolution of resistance, the myelomas all adopted adherent growth, independent of the morphology of the parent cell lines. When adherent versions of the parent cell lines were derived without exposure to bortezomib, they were, surprisingly, resistant to bortezomib. All bortezomib-resistant myeloma sublines downregulated XBP-1 mRNA levels and the reduction was correlated with the fold resistance. Moreover, splicing of XBP-1 mRNA to the active form was also reduced. The adherent versions of the parent cell lines that showed de novo resistance to bortezomib also had lower levels of XBP-1. Bortezomib normally induces XBP-1 expression and splicing acutely within 12–24 hours (AH Lee, et al., 2003. PNAS 100: 9946) but the resistant sublines were less responsive than the sensitive parents in this respect. Protein levels of other components of the UPR were evaluated by immunoblotting; BiP, phosphorylated-eIF2α, ATF6α and p58IPK were found to be unchanged in the resistant sublines. Cytoplasmic and secreted immunoglobulins were assayed by immunoblotting and ELISA respectively but no differences between the parent and resistant sublines were observed. In a small cohort of relapsed/refractory patients whose bone marrow biopsy was collected prior to bortezomib treatment, the quantity of intracytoplasmic light chain in the myeloma cells, as detected by flow cytometry, did not predict clinical response. The bortezomib-resistant myeloma sublines showed significant cross-resistance to doxorubicin and, to a lesser extent, vincristine, but none to melphalan. The cross-resistance is associated with adherent growth, as it was also observed in the adherent versions of the parent cell lines which were never exposed to bortezomib. Moreover, functional analysis of the multi-drug transporter activity, by drug accumulation assay, showed no difference between parent and resistant sub-lines. These results suggest that resistance to bortezomib is associated with downregulation of the IRE-XBP-1 pathway, but not necessarily other components of the UPR, consistent with observations by others concerning BiP (DT Rutkowski, et al., 2006. Plos Biology 4, e374). The association between adherence, bortezomib resistance and low XBP-1 requires further study.

In-Vitro Treatment with the AKT-Inhibitor GSK 690693 Induces Cell Death in Lymphoma Cell Lines and in Primary CLL Cells and Is Followed by Caspase-3 Activation and Cytochrome C Release.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1589-1589
Author(s):  
Dirk Winkler ◽  
Thorsten Zenz ◽  
Daniel Mertens ◽  
Annett Habermann ◽  
Hartmut Döhner ◽  
...  
Keyword(s):  
Cell Death ◽  
Cytochrome C ◽  
Conditioned Medium ◽  
Cell Lines ◽  
Caspase 3 ◽  
Spontaneous Apoptosis ◽  
Akt Inhibitor ◽  
Cytochrome C Release ◽  
Viable Cells

Abstract The PI3K/AKT pathway acts as a critical regulator of cell survival by stimulating cell proliferation and inhibiting apoptosis and has been implicated in the pathogenesis of lymphoproliferative disorders. Therefore, inhibition of AKT seems to be a highly attractive new approach for the treatment of lymphoma. We treated 9 cell lines with AKT-nhibitor (1, 10, 20 μM) over 24h and 48h respectively: EHEB (B-CLL), GRANTA-519 (MCL), JURKAT (T-ALL) BL-60, NAMALWA and BJAB (all Burkitt’s lymphoma), L363, OPM-2 and RPMI-8226 (all multiple myeloma). To determine the rates and type of AKT-inhibitor induced cell death, FACS analyses for CD19, 7AAD, active caspase-3, cytochrome c were performed. The phosphorylation status of AKT and its downstream proteins GSK3β, p70S6k and S6 was studied by Western blotting after 5–120 minutes. In addition, 11 primary CLL samples with either del 13q (n=3), del 11q (n=2), del 17p (n=3) or a normal karyotype (n=3) were treated with AKT inhibitor (10 μM; 2.5μM; 0.625 μM; 0.156 μM). CLL samples were cultured in both standard medium as well as in HS5-(human stromal cells) conditioned medium to reduce spontaneous apoptosis of CLL in-vitro. 6 out of 11 patients had unmutated VH genes. 8 Patients were untreated, 3 were previously treated. Fludarabine (0.1 μM) was added to AKT-inhibitor in 11 cases to test for synergistic effects. CLL cells were harvested after 48 hours and 5 days to measure cell viability using Celltiter-GLO-Assay. Treatment of cell lines lead to significant rates of AKT-inhibitor induced cell death (table 1), to hyperphosphorylation of AKT and to inhibition of phosphorylation of GSK3β (after 5 min) and S6 (after 20 min) in all cell lines and of p70S6k (after 120 min) in GRANTA, JURKAT, NAMALWA and BJAB. Cell death did not depend on functional p53 gene. Treatment of primary CLL samples with AKT-inhibitor alone was followed by a decrease of cell viability in a time and concentration dependent manner regardless of the medium used (table 2). Only with the lowest concentration and when cultured in HS5-conditioned medium, no further reduction of viable cells was seen between 48h and 5d. Treatment with AKT-inhibitor as a single agent seemed to be at least as effective as treatment with fludarabine. Response was independent of the genetic subgroup, VH mutation status or prior treatment. High risk cases with del 17p responded worse to fludarabine alone when compared to cases without del 17p (i.e. 75% of viable cells after 5d at 10000 μM in cases with del 17p vs. 25% in cases without del 17p). The same fludarabine resistant cases showed good responses to treatment with AKT-inhibitor (9% of viable cells after 5d at 10000 μM in cases with del 17p). A synergistic effect was not achieved by combining AKT-inhibitor and fludarabine. Culture of CLL cells in HS5-conditioned medium resulted in lower rates of spontaneous apoptosis, but also in lower rates of AKT-inhibitor induced cell death. In conclusion, in-vitro treatment with AKT-inhibitor resulted in significant rates of cell death in cell lines and primary CLL cells, even in patients with del 17p or resistance to fludarabine. In cell lines, treatment with AKT-inhibitor was followed by typical features of apoptosis such as activation of caspase-3 and cytochrome c release. In CLL samples, prior treatment did not affect in-vitro response rates. These data underline the involvement of the PI3K/Akt pathway in the pathogenesis of lymphoma and point to an efficacy of the AKT-inhibitor in the treatment of lymphoma, multiple myeloma and CLL in-vivo. Concerning CLL, the AKT-inhibitor seems to be an attractive new treatment option even for cases with high risk cytogenetics. Using HS5-conditioned medium seems to be a well functioning method to reduce spontaneous apoptosis of CLL cells in-vitro. Table 1: rates of cell death, caspase-3 activation and cytochrome c release after treatment of cell lines with AKT inhibitor (1μM, 48h) 7AAD-positive cells active caspase-3 cytochrome c release EHEB 15% − + GRANTA-519 15% + + JURKAT 17% + + BL60 24% + + NAMALWA 25% − (+) BJAB 30% + (+) L363 15% + − OPM-2 41% + + RPMI-8226 32% + (+) Table 2: mean percentage of viable cells after treatment with AKT-Inhibitor (A), fludarabine (F; 0,1μM) and their combination (A + F) measured by Celltiter-GLO-Assay 10000 nM 2500 nM 625 nM 156,25 nM 48h 5d 48h 5d 48h 5d 48h 5d A F A + F A F A + F A A F A+ F A F A+ F A HS5 + (n=8) 94 84 (n=5) 75 (n=5) 45 22 (n=5) 25 (n=5) 88 52 91 84 80 69 22 18 76 85 HS5 − (n=11) 60 79 59 8 39 21 77 27 80 79 76 28 39 34 82 (n=10) 21

Melphalan and XPO1 Inhibitor Combination Therapy for the Treatment of Multiple Myeloma

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2084-2084 ◽  
Author(s):  
Joel G Turner ◽  
Jana L Dawson ◽  
Steven Grant ◽  
Kenneth H. Shain ◽  
Yun Dai ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
De Novo ◽  
Ex Vivo ◽  
Myeloma Cell ◽  
Drug Resistant ◽  
Cell Viability Assay ◽  
Myeloma Cells ◽  
Multiple Myeloma Cell

Abstract Introduction High-dose melphalan chemotherapy with autologous stem cell transplant remains the standard of care for the treatment of multiple myeloma. However, patients eventually develop drug resistance and die from progressive disease despite the introduction of therapies using proteosome inhibitors (PIs) and immunomodulatory drugs (IMIDs). The incurable nature of multiple myeloma clearly demonstrates the need for novel agents and treatments. Here, our aim was to investigate whether the use of XPO1 (exportin 1, CRM1) inhibitors (XPO1i) could sensitize de novo and acquired drug-resistant multiple myeloma cells both in vitro and ex vivo to the alkylating agent melphalan. Materials and Methods Human multiple myeloma cell lines NCI-H929, RPMI-8226, U266 and PBMC controls were treated in vitro with the XPO1i KOS-2464 and the orally available Selective Inhibitor of Nuclear Export (SINE) selinexor (KPT-330) or) +/- melphalan. Multiple myeloma cells were grown at high-density conditions (>3-5x106 cells/mL). High-density multiple myeloma cells have been shown to possess de novo drug resistance. Sensitivity of the XPO1i/melphalan-treated NCI-H929 cells was measured by cell viability assay (CellTiter-Blue). Apoptosis in XPO1i/melphalan-treated NCI-H929, RPMI-8226, and U266 cells was assayed using flow cytometry (activated caspase 3). Proximity ligation assays were performed to assess XPO1-p53 binding in the presence of an XPO1i. Western blots of XPO1i-treated myeloma cells were performed for nuclear and total p53. Drug-resistant U266 (PSR) and 8226 (8226/B25) myeloma cell lines were developed by incremental exposure to bortezomib. PSR cells are able to grow in 15 nM bortezomib and the 8226/B25 in 25 nM. These resistant myeloma cells were treated in vitro with XPO1i +/- melphalan. Sensitivity to therapy was measured by apoptosis and cell viability assay. Multiple myeloma cells isolated from patients with newly diagnosed, relapsed, or refractory disease were treated with XPO1i +/- melphalan and CD138+/light chain+ myeloma cells and assayed for apoptosis. Results Multiple myeloma cell (NCI-H929) viability was decreased synergistically by XPO1i when used in combination with melphalan, as shown by the calculated combinatorial index (CI) values. We examined sequencing of the drugs and found that concurrent treatment with melphalan (10 µM) and selinexor (300 nM) for 48 hours produced the best results (CI value 0.370, n=6). Sequential treatment (selinexor for 24 hours followed by melphalan for an additional 24 hours) or the reverse sequence had slightly less synergy, with CI values of 0.491 (n=9) and 0.565 (n=3), respectively. Normal PBMC control cells were unaffected by XPO1i/melphalan treatment as shown by viability and apoptotic assays. Proximity ligation assay demonstrated that XPO1i blocks XPO1/p53 binding. Western blot showed that the XPO1i treatment of myeloma cells increased nuclear and total p53. Drug-resistant 8226/B25 myeloma cells but not PSR cells were found to be resistant to melphalan when compared to parental cell lines. Both resistant myeloma cell lines were sensitized by XPO1i to melphalan as shown by apoptosis assay (3- to 10-fold). CD138+/light chain+ myeloma cells derived from newly diagnosed, relapsed, and refractory myeloma patients were also sensitized by XPO1 inhibitors to melphalan as demonstrated by apoptotic assays (e.g. activated caspase 3). Conclusions XPO1i synergistically improved the response of de novo and acquired drug-resistant myeloma cells to melphalan in vitro and ex vivo. It is possible that this synergy may be due to an increase of nuclear p53 by XPO1i and the reported activation of p53 by melphalan. Future studies include in vitro experiments using drug-resistant human U266 myeloma cells in NOD-SCID-gamma mice and clinical trials using melphalan in combination with the SINE selinexor. Combination therapies using selinexor and melphalan may significantly improve the treatment of myeloma. Disclosures Kauffman: Karyopharm Therapeutics: Employment. Shacham:Karyopharm Therapeutics: Employment.

Combination of Akt/PKB Inhibition (Perifosine) and Farnesyl Transferase Inhibition (Tipifarnib) Results in Increased Cell Death in Myeloma Cells Lines.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1568-1568 ◽  
Author(s):  
Rajni Sinha ◽  
Ebenezer David ◽  
Emily Zeilter ◽  
Claire Torre ◽  
Jonathan L. Kaufman ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Proliferation ◽  
Cell Death ◽  
Combination Therapy ◽  
Cell Line ◽  
Cell Lines ◽  
Single Agent ◽  
Myeloma Cell ◽  
Myeloma Cell Line

Abstract Introduction Multiple myeloma is a clonal plasma cell malignancy characterized by proliferation and accumulation of plasma cells in the bone marrow. Most patients are incurable with the current treatment modalities. Clearly novel agents are needed to improve the outcome for patients with myeloma. We have previously shown that the combination of bortezomib and tipifarnib results in synergistic myeloma cell death. This increase in apoptosis is associated with down regulation of phosphorylated AKT, a potent anti-apoptotic signaling molecule. Therefore, agents that target AKT represent ideal compounds for further study in myeloma. Perifosine is a novel, oral bioavailable alkylphospholipid. Perifosine has displayed apoptotic and antipropliferative activity in vitro and in vivo in several human cancer models including leukemia. Perifosine exerts its actions by interfering with key intracellular pathways including AKT, MAPK, JNK, p21waf1. Our hypothesis is that targeting AKT via multiple upstream pathways will result in increased myeloma cell apoptosis. Therefore, we assessed the effects of single agent perifosine with and without tipifarnib on multiple myeloma cell lines. Method The myeloma cell line RPMI8226 was used. Cell viability and proliferation were assessed using MTT assays. Cells were incubated with increasing concentrations of both agents alone and in combination. Cell proliferation was assayed at 24, 48 and 72 hours. Western blots were then carried out to evaluate the effects of the intracellular protein PDK1, one of the critical signaling molecules that phosphorylates and activates AKT. Results As we and others have previously shown, tipifarnib at concentrations that can be achieved clinically is associated with minimal cytotoxicity. At 5 μM, tipifarnib decrease proliferation by only 20%. In contrast, there is a potent dose response effect of single agent perifosine (Fig. 1). These results were apparent as early as 24 hours. When tipifarnib at 5 μM is used in combination with a subtherapeutic dose of perifosine (2 μM), there is a marked decrease in cell proliferation (Fig. 2). In addition, combination therapy resulted in a reduction in the phosphorylated form of PDK1, a critical finding that was not seen with either drug alone. Conclusion Combination therapy with tipifarnib and perifosine results in less cell proliferation compared to either agent used alone in the RPMI8226 myeloma cell line. The dosages employed in these in-vitro studies are lower than those used in previously published data and are clinically achievable. Studies targeting other cell lines including MM.1R, MM.1S, and U266 are in progress. Analysis of AKT, Caspase 3, 8 and 9 are being explored to help delineate the mechanism of this novel combination. The goal is to develop further effective treatment options for patients with myeloma. Figure 1 Figure 1. Figure 2 Figure 2.

BH3 Profiling Predicts On-Target Cell Death Due To Selective Inhibition Of BCL-2 By ABT-199 In Acute Myelogenous Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 238-238 ◽  
Author(s):  
Leah Hogdal ◽  
Daniel J. DeAngelo ◽  
Richard M. Stone ◽  
Verena I. Gaidzik ◽  
Donna Bucci ◽  
...  
Keyword(s):  
Cell Death ◽  
Cytochrome C ◽  
Cell Lines ◽  
Ex Vivo ◽  
Functional Dependence ◽  
Predictive Biomarker ◽  
Selective Inhibition ◽  
Myelogenous Leukemia ◽  
Mitochondrial Activity ◽  
Cytochrome C Release

Abstract Most patients with acute myeloid leukemia (AML) become resistant to chemotherapy at some point in their course and succumb to their disease. It is necessary to prevent chemo-resistance or enhance chemosensitivity in a selective fashion to lead to a higher cure rate and a lower toxic burden. The B-cell leukemia /lymphoma 2 (BCL-2) protein acts to prevent commitment to programmed cell death initiated in the mitochondrion. Inhibiting the function of this protein is an attractive approach to cancer therapy, but it remains a challenge to identify those tumors that will best be treated with such a strategy. Here, we test the sensitivity of AML cell lines and primary patient samples to the selective BCL-2 inhibitor, ABT-199, and test for correlation between ABT-199 sensitivity with BCL-2 dependence measured by BH3 profiling. We hypothesized that cells that are more dependent on BCL-2 will be more sensitive to inhibition by ABT-199. To this end, we tested the sensitivity of seven AML cell lines and 34 primary patient samples to ABT-199. We found that both cell lines and primary patient samples were sensitive to ABT-199 (Figure 1). In the case of primary patient cells, the median EC-50 was approximately 20 nM, and cell death could be seen within 2 hours. Importantly, we found that sensitivity to ABT-199 was independent of cytogenetics and NPM1 and FLT3 mutations of the primary patient cells suggesting that treatment with ABT-199 could be useful for a variety of AML patients, even those with an intrinsically poor prognosis. We also measured dependence on BCL-2-mediated apoptosis in cell lines and primary patient samples by BH3 profiling. BH3-profiling is a method to determine the mitochondrial priming level of a cell by exposing cellular mitochondria to standardized amounts of peptides derived from the BH3 domains of BH3-only proteins and determining the rate of cytochrome c release. BH3-profiling can also identify which anti-apoptotic species are critical in mediating cell death in a given cell type. For instance, the BAD BH3-only protein binds with high affinity to BCL-2, BCL-XL and BCL-W. Thus, release of cytochrome c following BAD peptide incubation suggests an anti-apoptotic dependency on BCL-2, BCL-XL or BCL-W. Therefore, we tested whether ABT-199 sensitivity correlates with functional dependence on BCL-2 in both cell lines and primary patient samples. We found that cells that released cytochrome c following incubation with the BAD peptide were more sensitive to ABT-199 treatment (Figure 2). This demonstrates that ABT-199 functions on target at the mitochondria since the ABT-199 mitochondrial activity correlates well with ABT-199 cytotoxicity data. Since ABT-199 does not inhibit MCL-1, increased expression of MCL-1 could be a potential source of upfront resistance to BCL-2 inhibition. Therefore, we asked if there was a correlation between MCL-1 dependence and ABT-199 sensitivity. We observed a weak anti-correlation between mitochondrial sensitivity to ABT-199 and sensitivity to the MCL-1 selective peptide NOXA BH3. This suggests that there is a minor tendency for MCL-1 dependent mitochondria to be less sensitive to ABT-199. The ex-vivo sensitivity of AML cells to ABT-199, which appears to be BCL-2 specific, is similar to that observed in CLL, a disease for which ABT-199 has demonstrated consistent activity in clinical trials. Moreover, the BH3 profiling studies demonstrate ABT-199 activity at the mitochondrion that correlates very well with cytotoxicity, supporting a mitochondrial mechanism of action. Our BH3 profiling studies will be undertaken to determine if the results will serve as a predictive biomarker in an upcoming phase II clinical trial of ABT-199 in AML. Disclosures: Letai: AbbVie: Consultancy; Dana-Farber Cancer Institute: Patents & Royalties.

Dexamethasone Synergizes with ABT-199 through the Induction of Bim and Bcl-2 Dependence in Myeloma

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3447-3447
Author(s):  
Shannon Matulis ◽  
Ajay K. Nooka ◽  
Hayley Von Hollen ◽  
Jonathan L. Kaufman ◽  
Sagar Lonial ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Survival Rate ◽  
Cell Lines ◽  
Combination Treatment ◽  
Research Funding ◽  
Single Agent ◽  
Myeloma Cell ◽  
Fold Change ◽  
Binding Pattern ◽  
Apoptotic Protein

Abstract Multiple myeloma is a plasma cell malignancy, with a 10-year survival rate of approximately 25%. While this survival rate is largely attributed to the FDA approval of new therapies for the disease, such as IMiDs and proteasome inhibitors, eventual relapse and drug resistance is the reality facing the majority of myeloma patients. Subsequently, the quest for new drugs and combination therapies is ongoing. Novel agents targeting the Bcl-2 family of cell survival regulators are promising avenues of research. We have previously reported on a method of predicting sensitivity of myeloma cell lines and patient samples to the Bcl-2/xL inhibitor ABT-737, based on the binding pattern of pro-apoptotic protein Bim to anti-apoptotic proteins Mcl-1, Bcl-xL, and Bcl-2. In Mcl-1-dependent cells, Bim is primarily associated with Mcl-1, and insensitive to ABT-737. Alternatively, in cells that are co-dependent on Mcl-1 and Bcl-2/xL for survival, Bim is either predominantly associated with Bcl-2/xL or when it is released from Bcl-2/xL it cannot bind to Mcl-1 because of the presence of the Mcl-1 inhibitor Noxa, rendering the cells sensitive to ABT-737. Unfortunately, ABT-737, or the clinical compound Navitoclax, is not a viable treatment for myeloma, due to its potential for causing thrombocytopenia. Therefore we chose to investigate the sensitivity of myeloma cell lines and freshly isolated patient samples to ABT-199. ABT-199, a specific inhibitor of anti-apoptotic protein Bcl-2, is currently in phase I clinical trials for multiple myeloma. We have previously reported on its preclinical efficacy as a single agent in myeloma as well as in combination with other commonly used therapeutics. While ABT-199 was ineffective as a single agent in the cell lines we tested, combining it with dexamethasone significantly decreased the IC50s (Fold change: 8226 – 2.6, MM.1s – 43.5, OPM2 – 14.1, KMS11 – 23.3, KMS18 – 4.8). Here we report on the mechanism by which dexamethasone increases sensitivity to ABT-199, as well as the effectiveness of this combination in freshly isolated patient samples. We performed real-time PCR analysis to determine changes in the expression of the Bcl-2 family of proteins following treatment with ABT-199, dexamethasone, and the combination. Minimal changes in expression were seen with ABT-199 treatment; however, dexamethasone treatment greatly induced the expression of both Bcl-2 and Bim, which was also seen in the combination treatment, along with a decrease in Bcl-xL expression. Next we performed CoIP studies to examine how these changes in expression affected the binding pattern of Bim to the anti-apoptotic proteins. As expected, we found that upon treatment with ABT-199, the small amount of Bim previously bound to Bcl-2 was released. Alternatively, when cells were treated with dexamethasone alone, the amount of Bim bound to Bcl-2 increased, likely due to the significant increase in expression of both proteins. When cells were subjected to both drugs simultaneously, there was a decrease in the amount of Bim bound to Bcl-xL, as well as an absence of Bim on Bcl-2. Thus, co-treatment brings about a scenario whereby ABT-199 releases Bim bound to Bcl-2, and at the same time, prevents the Bim induced by dexamethasone from binding to Bcl-2. These changes, coupled with a decrease in amount of Bcl-xL available to bind Bim, result in the induction of apoptosis. To further verify this drug synergy, we treated ficoll-isolated buffy coat cells from 10 myeloma patients with ABT-199, either alone or in combination with dexamethasone. Samples from 4 of the patients were sensitive to ABT-199 alone, thus combination had little effect. For the other 6 patient samples, the addition of dexamethasone to ABT-199 significantly decreased the IC50 over ABT-199 alone (Fold Change: MM61 – 7.3, MM62 – 17.5, MM63 – 20.1, MM64 – 2.2, MM65 – 1.8, MM66 – 8.7). Previous studies have reported that the t(11;14) subset of multiple myeloma is highly sensitive to ABT-199. Five of the 10 patient samples we tested were positive for the t(11;14), and all but one, MM61, were sensitive to ABT-199 alone. However, MM61 was sensitive to the combination treatment. Taken together, our data demonstrate that the addition of dexamethasone expands the potential of ABT-199 to a broader set of patients who would otherwise likely be resistant to monotherapy. Disclosures Kaufman: Millennium: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Onyx: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria; Spectrum: Consultancy, Honoraria; Merck: Research Funding. Lonial:Millennium: The Takeda Oncology Company: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Onyx Pharmaceuticals: Consultancy, Research Funding.

scholarly journals Chaetocin: a promising new antimyeloma agent with in vitro and in vivo activity mediated via imposition of oxidative stress

Blood ◽  
2006 ◽  
Vol 109 (6) ◽  
pp. 2579-2588 ◽  
Author(s):  
Crescent R. Isham ◽  
Jennifer D. Tibodeau ◽  
Wendy Jin ◽  
Ruifang Xu ◽  
Michael M. Timm ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
B Cells ◽  
Cell Lines ◽  
Ex Vivo ◽  
Myeloma Cell ◽  
Lymphocytic Leukemia ◽  
Myeloma Cells ◽  
High Degree

Abstract Chaetocin, a thiodioxopiperazine natural product previously unreported to have anticancer effects, was found to have potent antimyeloma activity in IL-6–dependent and –independent myeloma cell lines in freshly collected sorted and unsorted patient CD138+ myeloma cells and in vivo. Chaetocin largely spares matched normal CD138− patient bone marrow leukocytes, normal B cells, and neoplastic B-CLL (chronic lymphocytic leukemia) cells, indicating a high degree of selectivity even in closely lineage-related B cells. Furthermore, chaetocin displays superior ex vivo antimyeloma activity and selectivity than doxorubicin and dexamethasone, and dexamethasone- or doxorubicin-resistant myeloma cell lines are largely non–cross-resistant to chaetocin. Mechanistically, chaetocin is dramatically accumulated in cancer cells via a process inhibited by glutathione and requiring intact/unreduced disulfides for uptake. Once inside the cell, its anticancer activity appears mediated primarily through the imposition of oxidative stress and consequent apoptosis induction. Moreover, the selective antimyeloma effects of chaetocin appear not to reflect differential intracellular accumulation of chaetocin but, instead, heightened sensitivity of myeloma cells to the cytotoxic effects of imposed oxidative stress. Considered collectively, chaetocin appears to represent a promising agent for further study as a potential antimyeloma therapeutic.

Inhibition Of Wee1 Enhances The Anti-Leukemic Effects Of Antimetabolites In Vitro and In Vivo

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1281-1281
Author(s):  
James B. Ford ◽  
Susan Fosmire ◽  
Annemie van Linden ◽  
Dmitry Baturin ◽  
Christopher C. Porter
Keyword(s):  
Combination Therapy ◽  
Mouse Model ◽  
Acute Leukemia ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Single Agent ◽  
Parp Cleavage

Abstract While some patients with acute leukemia are cured, for many subsets of patients current therapeutic strategies are not adequate. Novel therapeutic approaches are needed for patients with higher risk leukemias, including T-ALL and AML. We and others identified Wee1 as a potential target in AML cells using RNAi screening. We have validated chemosensitization to cytarabine by genetic and pharmacologic inhibition of Wee1 in AML cell lines and primary patient samples ex vivo. A Wee1 inhibitor, MK1775, is in clinical development. We sought to further our findings with a wider range of conventional anti-leukemia agents, to determine whether the functionality of p53 influences chemosensitization, and to determine the tolerability and efficacy of MK1775 in combination with cytarabine in a mouse model of leukemia. We have found that MK1775 synergistically inhibits proliferation of the T-ALL Jurkat cell line with several antimetabolite chemotherapeutics including cytarabine, 6-thioguanine, and methotrexate. In contrast, MK1775 does not sensitize Jurkats to doxorubicin or etoposide, suggesting specific sensitization to antimetabolites. The addition of MK1775 enhances the antimetabolite induced apoptosis, as measured by Annexin V/7-AAD staining, and PARP cleavage measured by Western blotting. As expected, the addition of MK1775 enhances DNA damage induced by cytarabine as measured by γH2AX staining and flow cytometry, although preliminary data suggest that this is not the only mechanism of enhanced cell death, as a substantial proportion of cleaved PARP+ cells does not stain for γH2AX. In addition, we have found that AML cell lines with both wild-type and mutated TP53 are sensitive to chemosensitization by Wee1 inhibition. Furthermore, in isogenic models of p53 dysfunction, we have found that the functionality of p53 does not influence chemosensitization. Lastly, in an aggressive mouse model of AML, we observed enhanced disease control and survival in mice treated with MK1775 and ARA-C as compared to ARA-C alone. Hematotoxicity associated with treatment was related to the duration of combination therapy, but was tolerated well with intermittent dosing. Taken together, these data indicate that Wee1 inhibition may enhance the efficacy of several clinically relevant anti-leukemia agents, particularly the antimetabolites, but not topoisomerase inhibitors. Further, they suggest caution about the use of p53 mutation as a biomarker predictive of response to Wee1 inhibition. Moreover, we show that the addition of MK1775 to cytarabine is tolerable and more effective than cytarabine alone in vivo. Ongoing studies are aimed at better understanding the mechanism of combinatorial effect and to determine whether combination therapy is more efficacious than single agent therapy in xenograft models of leukemia. These data provide justification for early phase clinical trials of MK1775 in combination with antimetabolites in patients with high risk acute leukemia. Disclosures: No relevant conflicts of interest to declare.

The Hedgehog Pathway As Targetable Vulnerability with 5-Azacitidine in MDS and AML

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4612-4612
Author(s):  
Raoul Tibes ◽  
Devora Delman ◽  
Nanna Hansen ◽  
Krisstina L. Gowin ◽  
Ruben Mesa ◽  
...  
Keyword(s):  
Cell Lines ◽  
Ex Vivo ◽  
Single Agent ◽  
Myeloid Cells ◽  
Specific Effect ◽  
Drug Dose ◽  
Therapeutic Interventions ◽  
Chromosome 5 ◽  
Clonogenic Growth

Abstract Introduction/Background Recurrent deletions of chromosome 5 and 7 are characteristic of the myeloid malignancies MDS and AML, determine prognosis and influence therapeutic decision. The pathogenetic contribution of individual genes to MDS/AML development has been shown for several of these genes. However, how and if these genetic losses may relate to therapeutic interventions or represent haploinsufficient targets has remained elusive for the most part. We hypothesized that genes found within the commonly deleted regions (CDRs) of chromosome 5/7 (chr 5/7) represent targetable vulnerabilities in malignant myeloid cells. Therefore we generated a custom RNAi library to functionally interrogate these genomic regions alone or under 5-Azacitidine (5-Aza) treatment, to identify individual genes whose silencing modulate 5-Aza responsiveness. Methods/Materials Custom siRNA plates against ~270 genes (3x sequences/gene, Qiagen) on CDRs of chr 5/7 were assembled. Genes were silenced by siRNA for 48 hours followed by 48-72 hour 5-Aza treatment after which cell viability was determined. Four cell lines (TF1, THP1, MDS-L and HEL) were investigated in parallel with combined siRNA/5-Aza incubations and appropriate 5-Aza and siRNA only control. Hits were selected as > 2 standard deviation changes in viability from the median log2 value of the ratio [(siRNA + 5-Aza)/(siRNA only)], normalized per plate and across the entire screen. Duplictae RNAis screens were performed. Synergy between 5-Aza and the SMO inhibitors LDE225 (Sonidegib) and GDC0449 (Vismodegib) was assessed with CalcuSyn in a panel of AML cell lines. Ex vivo viability and clonogenic re-population experiments in primary patient derived AML and MDS cells were performed with Sonidegib. Results Several genes within the Hedgehog (HH) pathway emerged as sensitizers to 5-Aza when silenced by siRNA in three of four cell lines examined. Specifically Shh siRNA sensitized to 5-Aza in both TF-1 and THP-1 cells, SMO in HEL and GLI3 in THP-1 cells. Shh silencing alone (siRNA only) resulted in reduction of viability only in TF-1 cells but not in THP-1 cells, while SMO and GLI3 siRNA alone did not result in significant reductions in viability but sensitized to 5-Aza, indicating true sensitization and an interactions between the HH pathway and 5-Aza treatment. The SMO inhibitor Vismodegib synergized with 5-Aza in drug dose response assays in a panel of four AML cell lines (TF-1, THP-1, ML-2, HL-60) with Combination Index (CI) values of around 0.6 or lower at low doses of both drugs. Similar results were also observed with Sonidegib in vitro in 5 molecularly heterogeneous AML cell lines. Given stem cell modulation capacity of SMO inhibition, we examined ex vivo proliferation and more importantly clonogenic growth capacity. Highly interesting, ex vivo viability was reduced and synergy observed by the Sonidegib/5-Aza combination to a much greater extend in CD34+ selected primary MDS and AML cells as compared to bulk or CD34 depleted (normal) MNCs. Clonogenic growth was reduced in the combination over single agent 5-Aza or Sonidegib in ~ 50% of samples assessed to date. No direct correlations to cytogenetics were observed. A clinical Phase 1b trial was designed based on these results and is currently accruing in the first cohorts. Conclusions RNAi screening of CDRs of chr 5/7 yielded potential targetable therapeutic vulnerabilities with several genes within the HH pathway sensitizing to 5-Aza. SMO inhibitors pharmacologically validate this concept in vitro and ex vivo with the potential to more selectively affect leukemia stem/progenitor cells. Clinical data will show if this is a specific effect involving chr 5/7 or a general concept in malignant myeloid cells. Thus, SMO inhibition in combination with 5-Aza may be a novel rational combination in AML and MDS. Disclosures Off Label Use: LDE225/Sonidegib on a clinical trial. Mesa:Incyte, CTI, NS Pharma, Inc., Gilead, Celgene: Research Funding.

scholarly journals Bax-induced cytochrome c release from mitochondria depends on alpha-helices-5 and -6

2004 ◽  
Vol 378 (1) ◽  
pp. 247-255 ◽  
Author(s):  
Gerd HEIMLICH ◽  
Alastair D. McKINNON ◽  
Katussevani BERNARDO ◽  
Dieter BRDICZKA ◽  
John C. REED ◽  
...  
Keyword(s):  
Cytochrome C ◽  
Transmembrane Domain ◽  
Mitochondrial Outer Membrane ◽  
Deletion Mutants ◽  
Intermembrane Space ◽  
Mitochondrial Membranes ◽  
Cytochrome C Release ◽  
Alpha Helices ◽  
Bh3 Domain ◽  
Apoptotic Protein

The pro-apoptotic protein Bax plays a key role in the mitochondrial signalling pathway. Upon induction of apoptosis, Bax undergoes a conformational change and translocates to mitochondrial membranes, where it inserts and mediates the release of cytochrome c from the intermembrane space into the cytosol. However, the domains of Bax that are essential for the induction of cytochrome c release are still elusive. Therefore various Bax deletion mutants were generated and expressed in Escherichia coli. The proteins were then purified in order to delineate the function of the transmembrane domain, the BH3 (Bcl-2 homology 3) domain and the putative pore-forming α-helices-5 and -6. These proteins were used to analyse the mechanism of Bax-induced cytochrome c release from mitochondria. None of the Bax proteins caused cytochrome c release merely through physical perturbation of the mitochondrial outer membrane. The α-helices-5 and -6 of Bax were shown to mediate the insertion of the protein into mitochondrial membranes and to be essential for the cytochrome c-releasing activity of Bax. In contrast, neither the transmembrane domain nor a functional BH3 domain is required for the Bax-mediated release of cytochrome c from mitochondria.

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