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.

Strategies To Identify Effective Treatments For Proteasome Inhibitor Resistant Multiple Myeloma

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 278-278
Author(s):  
Brian Van Ness ◽  
Holly A. F. Stessman ◽  
Linda B. Baughn ◽  
Aatif Mansoor ◽  
Amit Mitra ◽  
...  
Keyword(s):  
Data Base ◽  
Cell Lines ◽  
Research Funding ◽  
Human Cancer ◽  
Expression Profiles ◽  
Single Agent ◽  
Cross Resistance ◽  
Human Cancer Cell Lines ◽  
Chemical Structures

Abstract Despite the introduction of effective new agents in the treatment of myeloma, the disease is still mostly incurable. Among the most significant issues is the heterogeneity of the disease, with accumulation of multiple genetic abnormalities among patients, resulting in disease refractory to some highly active agents, or the emergence of resistance leading to aggressive relapse. We have focused efforts on modeling drug sensitivity, and generating both genetic and biomarker signatures of response and resistance to the proteasome inhibitors (PIs): bortezomib (Btz), MLN2238 (Takeda) and carfilzomob (Onyx). Using mouse and human myeloma cell lines we dose escalated with bortezomib and selected bortezomib-resistant (Btz-R) lines (that showed cross resistance to MLN2238 and carfilzomib). We previously reported the identification of gene expression signatures that distinguish sensitivity or resistance to bortezomib (Stessman, et al., Mol Cancer Ther 12:1140, 2013). We then took three approaches to identify effective strategies to treat bortezomib resistance: 1) The connectivity map (CMAP) data base contains treatment induced transcriptional signatures from over 1300 bioactive compounds in human cancer cell lines. We queried our Btz-R expression profiles against the CMAP data base and developed a correlation analysis to identify potential drugs that would be predicted to show toxicity to Btz-R lines. We identified HDAC-inhibitor and topoisomerase-inhibitor sensitivity profiles that were able to predict Btz-R tumor responsiveness to these drugs in vitro. 2) We found that Btz-R was associated with loss of PC maturation markers (CD69, CD93, BLIMP, CXCR4, spliced XBP-1), and were able to re-sensitize Btz-R cells to Btz sensitivity by inducing PC maturation. The correlation of expression markers we identified stratified survival outcomes in bortezomib containing clinical trials; thus demonstrating the markers we identified in the in vitro modeling are relevant to patient outcomes. 3) Finally, we designed a cell-based high throughput drug screening approach that led to the identification of several chemical structures with selective activity against Btz-R cells. Topoisomerase-inhibitors were among the top hits, a finding that independently supported our CMAP screening results. We also identified a more novel chemical lead (Velcade Re-sensitizing Compound 2; VRC-2) that showed modest but significant selectivity for Btz-R cells as a single agent, and most notably, exhibited potent Btz re-sensitizing activity when the 2 drugs were combined. The Btz re-sensitizing activity of VRC-2 was confirmed using multiple human and mouse Btz-R cell lines. The initial successes of the in vitro approaches that will be presented demonstrate the value of profiling resistant tumors as a means to identify secondary therapies, and demonstrate there are powerful screening approaches that can be used to effectively treat or reverse drug resistance. Disclosures: Van Ness: Millenium Pharnaceutical: Honoraria, Research Funding; Onyx Pharmaceutical: Research Funding.

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.

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.

Anti-Myeloma Activity of Selective PI-3K/PDK/mTOR Inhibitor BEZ235.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1185-1185 ◽  
Author(s):  
Douglas W. McMillin ◽  
Joseph Negri ◽  
Jake Delmore ◽  
Patrick Hayden ◽  
Nicholas Mitsiades ◽  
...  
Keyword(s):  
Cell Lines ◽  
Mtor Inhibitor ◽  
Mononuclear Cells ◽  
Single Agent ◽  
In Vivo Studies ◽  
Cross Resistance ◽  
Bone Lesions ◽  
Normal Donor

Abstract Context: The PI3K-Akt-mTOR pathway has been a promising target for the treatment of multiple myeloma (MM). Major cytokine/growth factor receptor cascades (e.g. IGF-1/IGF-1R or IL-6/IL-6R) mediate, at least in part, their proliferative, anti-apoptotic or drug resistance effects through PI3K-Akt-mTOR activation and their downstream effectors. Therefore blocking this signaling pathway at one, or preferably more, of its molecular levels is considered to have promising therapeutic potential for MM. The small molecular mass compound NVP-BEZ235 (Novartis Pharma, Basel Switzerland) allows a multi-targeted, yet selective, inhibition of the PI-3K/Akt/mTOR signaling axis at the level of PI-3K and mTOR and was tested in our pre-clinical MM models. Methods/Results: A panel of human MM cell lines was tested for their in vitro response to NVP-BEZ235 using MTT colorimetric survival assays. All MM cell lines tested exhibited dose- and time-dependent decrease of their viability upon exposure to NVP-BEZ235 (IC50= 25–800 nM for 24–48hrs), without evidence of potential cross-resistance between conventional or novel anti-MM agents and NVP-BEZ235. Indeed, MM cells highly sensitive (IC50 <25 nM) to NVP-BEZ235 (e.g. MM.1S, MM.1R, Dox40 and KMS-12-PE) included both lines known to be sensitive as well as others which are resistant to dexamethasone, cytotoxic chemotherapy, thalidomide and/or its immunomodulatory derivatives (IMIDs). A longitudinal assessment of viability of MM-1S and OPM-2 MM cells during a 48-hr incubation with pharmacologically relevant concentrations of NVP-BEZ235 (25– 400nM) showed rapid commitment to and induction of MM cell death. This result, coupled with the observation that normal donor peripheral blood mononuclear cells (PBMCs) were less sensitive (IC50 >800 nM) than all MM cell lines tested, suggest that this compound exhibits a rapid and tumor-selective effect at clinically relevant conditions. This observation is further supported by our preliminary in vivo studies which suggest anti-MM activity of the drug in a model of diffuse MM bone lesions in SCID/NOD mice. Optimization of dosing and schedule to improve overall survival of NVP-BEZ235 treated mice is ongoing. To provide a more comprehensive framework for possible clinical applications of NVP-BEZ235 for MM treatment, we evaluated a series of combinations of this agent with conventional (e.g. dexamethasone, doxorubicin) and novel (e.g. bortezomib, immunodulatory thalidomide derivatives) anti-MM agents. Given the very potent single-agent activity of NVP-BEZ235 at even low nM concentrations, formal statistical documentation of synergy was not observed, but encouragingly no evidence of antagonism with any of these anti-MM agents was observed, indicating that combinations of NVP-BEZ235 with the aforementioned anti-MM agents can be feasible in clinical settings. Conclusion: The dual PI3K/mTOR inhibitor NVP-BEZ235 induces MM cell killing at sub-μM concentrations, with significantly higher sensitivity of MM cells compared to normal tissues, suggesting that this kinase inhibitor merits further consideration for possible testing as treatment option for MM patients. Further in vitro and in vivo studies are ongoing to further support the translation of these observations to clinical trials in MM.

IL-6-Induced C-MYC Down-Regulates the Expression of CD33 in CD33(+) Myeloma Cell Lines.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2496-2496
Author(s):  
Karim Shamsasenjan ◽  
Ken-ichiro Otsuyama ◽  
Mohd S. Iqbal ◽  
Saeid Abroun ◽  
Jakia Amin ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Ectopic Expression ◽  
Cell Lineage ◽  
Myeloma Cell ◽  
Mrna Levels ◽  
Down Regulation ◽  
Myeloma Cells ◽  
Lineage Markers

Abstract Since human myeloma cells specifically lose the expression of PAX-5 gene, the master gene of B cell lineage, we frequently detect the ectopic expressions of non-B cell lineage markers such as CD56 and CD33. As for the expression of CD33, we confirmed that CD33 expression was found in primary myeloma cells from about 12% of 120 cases with overt myeloma and also in 2 myeloma cell lines (ILKM8 and Liu01 (a subclone of U266)) in the protein and mRNA levels. In primary myeloma cells as well as CD33(–) myeloma cell lines, DMSO treatment could induce the expression of CD33 in the in vitro culture. In these CD33(+) myeloma cell lines and DMSO-induced myeloma cells, we found that expressions of C/EBPα and PU.1 were markedly increased by gene expression profiling. On the other hand, IL-6 down-regulated the expression of CD33 in CD33(+) myeloma cell lines accompanying with down-regulation of C/EBPα and PU.1 expressions. Also, we found that IL-6 up-regulated the expression of C-MYC and the increased C-MYC bound to the promoter region of C/EBPα gene followed by the down-regulation of C/EBPα expression. It was confirmed that introduction of sh-RNA for C-MYC to two CD33(+) myeloma cell lines blocked the IL-6-induced down-regulation of CD33 and C/EBPα expression. Therefore, these results indicate that IL-6 can reverse the ectopic expression of non-B cell lineage markers by up-regulating C-MYC followed by its down-regulation of C/EBPα expression and also suggest that constitutive activation of STAT3 by IL-6 may keep PAX-5(–) myeloma cells being uncommitted cells to any lineage; myeloma cells could be one of stem cell-like cancer cells.

scholarly journals Combination of AZD4573, a Selective CDK9 Inhibitor, with Other Cell Death Inducing Agents Can Overcome De Novo Venetoclax Resistance in Preclinical Hematologic Tumor Models

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3946-3946
Author(s):  
Justin Cidado ◽  
Scott Boiko ◽  
Theresa Proia ◽  
Haley Woods ◽  
Omid Tavana ◽  
...  
Keyword(s):  
Cell Death ◽  
Tumor Growth ◽  
Growth Inhibition ◽  
Cell Lines ◽  
De Novo ◽  
Single Agent ◽  
Acquired Resistance ◽  
Tumor Xenograft ◽  
Tumor Growth Inhibition

Abstract Apoptosis is controlled through the dynamic interactions of the Bcl2 protein family, and cancers have evolved mechanisms to hijack this pathway to evade apoptosis, often by upregulating anti-apoptotic proteins (e.g. Mcl1, Bcl2). This survival adaptation creates a dependency that could be exploited therapeutically, which is why considerable effort has been made to develop small molecule inhibitors of the anti-apoptotic Bcl2 family proteins. This class of drug was clinically validated with the approval of venetoclax, a selective Bcl2 inhibitor, for the treatment of CLL. Venetoclax is undergoing evaluation in numerous other clinical trials for predominantly hematologic malignancies. Despite impressive responses observed with venetoclax in CLL (ORR 79%), acquired resistance is beginning to emerge. Likewise, other hematologic indications are more intrinsically resistant to venetoclax, exhibiting much lower response rates in the respective Phase I clinical trials (AML = 38%, NHL = 44%, MM = 12%). Preclinical studies with venetoclax have reported increased levels of other anti-apoptotic proteins as a likely mechanism contributing to both de novo and acquired resistance. Therefore, combining cell death inducing agents that inhibit Mcl1 or BclxL could be a means of combating resistance. Inhibition of cyclin-dependent kinase 9 (CDK9), which regulates transcription elongation, has been reported to reduce protein levels of genes with short-lived transcripts and proteins, such as MCL1. AZD4573 is a novel and selective CDK9 inhibitor that shows potent single agent activity, inducing cell death in vitro and tumor regressions in vivo in a diverse set of hematologic cancers (Cidado et. al., AACR Annual Meeting 2018). AZD4573 is currently being evaluated in a Phase I clinical trial for patients with hematological malignancies (NCT03263637). This study evaluates whether combinations of AZD4573 with other cell death inducing agents could overcome de novo venetoclax monotherapy resistance. A panel of 12 AML and 6 DLBCL cell lines were treated for 6 h with venetoclax, AZD4573, and a selective BclxL probe compound (AZ'3202) either as single agents or in combinations and assayed for caspase activation. The Loewe model was used to calculate synergy scores to assess benefit over monotherapy, and combinations with scores >5 were deemed beneficial. Treatment with AZD4573+venetoclax and AZD4573+AZ'3202 resulted in beneficial combinations for 13/18 and 10/18 cell lines, respectively. On the other hand, venetoclax+AZ'3202 showed significant combination benefit in only two cell lines (NB4, SUDHL4), suggesting a primary dependency upon Mcl1 for most of these hematologic cancer cell lines. Interestingly, cell lines sensitive to single agent AZ'3202 (4 AML, 0 DLBCL) did not show any combination benefit when treated with AZD4573+venetoclax, highlighting the exquisite dependency of those four models upon BclxL and mutual exclusivity with Mcl1. Cell lines benefitting from the AZD4573+venetoclax combination tended to fall into one of two categories: having single agent activity to either agent that is enhanced by the combination or having no single agent activity but the combination shifts the cell line into a responder. SUDHL4 cells were sensitive to AZD4573 (caspase activation EC50 = 18 nM) but not venetoclax (EC50 = 476 nM) while OCI-AML3 was insensitive to both (EC50 > 30 µM). In vitro biomarker kinetic analysis revealed an increase in Mcl1 levels (~2-fold) after 3 h of venetoclax treatment that was abrogated upon combination treatment, providing a mechanistic rationale for the combination benefit. Furthermore, when tested in an OCI-AML3 tumor xenograft study in mice, AZD4573 or venetoclax monotherapy exhibited minimal tumor growth inhibition (44% and 16%, respectively) while the combination led to tumor regressions (64%) with minimal effect on body weight. In a SUDHL4 tumor xenograft study, venetoclax monotherapy displayed minimal tumor growth inhibition (25%), but intermittent dosing of AZD4573 exhibited 94% tumor growth inhibition. Still, combination therapy demonstrated a clear benefit as it led to complete tumor regressions with 6/8 mice remaining tumor-free until the end of study (150 days). Together, this work presents supporting evidence that combining cell death inducing agents would be effective at overcoming de novo or acquired resistance associated with monotherapy treatments. Disclosures Cidado: AstraZeneca: Employment, Equity Ownership. Boiko:AstraZeneca: Employment. Proia:AstraZeneca: Employment. Woods:AstraZeneca: Employment. Tavana:AstraZeneca: Employment. San Martin:AstraZeneca: Employment. Tron:AstraZeneca: Employment. Shao:AstraZeneca: Employment. Drew:AstraZeneca: Employment.

scholarly journals TRIM27 interacts with Iκbα to promote the growth of human renal cancer cells through regulating the NF-κB pathway

BMC Cancer ◽  
2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Chengwu Xiao ◽  
Wei Zhang ◽  
Meimian Hua ◽  
Huan Chen ◽  
Bin Yang ◽  
...  
Keyword(s):  
Cell Lines ◽  
Prognostic Indicator ◽  
The Cancer Genome Atlas ◽  
Mrna Levels ◽  
Loss Of Function ◽  
Protein Levels ◽  
Kaplan Meier ◽  
Cancer Genome Atlas

Abstract Background The tripartite motif (TRIM) family proteins exhibit oncogenic roles in various cancers. The roles of TRIM27, a member of the TRIM super family, in renal cell carcinoma (RCC) remained unexplored. In the current study, we aimed to investigate the clinical impact and roles of TRIM27 in the development of RCC. Methods The mRNA levels of TRIM27 and Kaplan–Meier survival of RCC were analyzed from The Cancer Genome Atlas database. Real-time PCR and Western blotting were used to measure the mRNA and protein levels of TRIM27 both in vivo and in vitro. siRNA and TRIM27 were exogenously overexpressed in RCC cell lines to manipulate TRIM27 expression. Results We discovered that TRIM27 was elevated in RCC patients, and the expression of TRIM27 was closely correlated with poor prognosis. The loss of function and gain of function results illustrated that TRIM27 promotes cell proliferation and inhibits apoptosis in RCC cell lines. Furthermore, TRIM27 expression was positively associated with NF-κB expression in patients with RCC. Blocking the activity of NF-κB attenuated the TRIM27-mediated enhancement of proliferation and inhibition of apoptosis. TRIM27 directly interacted with Iκbα, an inhibitor of NF-κB, to promote its ubiquitination, and the inhibitory effects of TRIM27 on Iκbα led to NF-κB activation. Conclusions Our results suggest that TRIM27 exhibits an oncogenic role in RCC by regulating NF-κB signaling. TRIM27 serves as a specific prognostic indicator for RCC, and strategies targeting the suppression of TRIM27 function may shed light on future therapeutic approaches.

scholarly journals YB-1 Is Altered in Pregnancy-Associated Disorders and Affects Trophoblast in Vitro Properties via Alternation of Multiple Molecular Traits

2021 ◽  
Vol 22 (13) ◽  
pp. 7226
Author(s):  
Violeta Stojanovska ◽  
Aneri Shah ◽  
Katja Woidacki ◽  
Florence Fischer ◽  
Mario Bauer ◽  
...  
Keyword(s):  
Cell Lines ◽  
Cancer Progression ◽  
Cell Function ◽  
Trophoblast Cell ◽  
Mrna Levels ◽  
Trophoblast Cells ◽  
Invasion And Migration ◽  
And Migration ◽  
Apoptosis And Inflammation

Cold shock Y-box binding protein-1 (YB-1) coordinates several molecular processes between the nucleus and the cytoplasm and plays a crucial role in cell function. Moreover, it is involved in cancer progression, invasion, and metastasis. As trophoblast cells share similar characteristics with cancer cells, we hypothesized that YB-1 might also be necessary for trophoblast functionality. In samples of patients with intrauterine growth restriction, YB-1 mRNA levels were decreased, while they were increased in preeclampsia and unchanged in spontaneous abortions when compared to normal pregnant controls. Studies with overexpression and downregulation of YB-1 were performed to assess the key trophoblast processes in two trophoblast cell lines HTR8/SVneo and JEG3. Overexpression of YB-1 or exposure of trophoblast cells to recombinant YB-1 caused enhanced proliferation, while knockdown of YB-1 lead to proliferative disadvantage in JEG3 or HTR8/SVneo cells. The invasion and migration properties were affected at different degrees among the trophoblast cell lines. Trophoblast expression of genes mediating migration, invasion, apoptosis, and inflammation was altered upon YB-1 downregulation. Moreover, IL-6 secretion was excessively increased in HTR8/SVneo. Ultimately, YB-1 directly binds to NF-κB enhancer mark in HTR8/SVneo cells. Our data show that YB-1 protein is important for trophoblast cell functioning and, when downregulated, leads to trophoblast disadvantage that at least in part is mediated by NF-κB.

Simvastatin Induces Death of Multiple Myeloma Cell Lines

2004 ◽  
Vol 52 (5) ◽  
pp. 335-344 ◽  
Author(s):  
Naomi Gronich ◽  
Liat Drucker ◽  
Hava Shapiro ◽  
Judith Radnay ◽  
Shai Yarkoni ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Cell Death ◽  
Cell Lines ◽  
Myeloma Cell ◽  
Cytoplasmic Calcium ◽  
Multiple Myeloma Cell ◽  
Cycle Arrest ◽  
Rpmi 8226

BackgroundAccumulating reports indicate that statins widely prescribed for hypercholesteromia have antineoplastic activity. We hypothesized that because statins inhibit farnesylation of Ras that is often mutated in multiple myeloma (MM), as well as the production of interleukin (IL)-6, a key cytokine in MM, they may have antiproliferative and/or proapoptotic effects in this malignancy.MethodsU266, RPMI 8226, and ARH77 were treated with simvastatin (0-30 μM) for 5 days. The following aspects were evaluated: viability (IC50), cell cycle, cell death, cytoplasmic calcium ion levels, supernatant IL-6 levels, and tyrosine kinase activity.ResultsExposure of all cell lines to simvastatin resulted in reduced viability with IC50s of 4.5 μM for ARH77, 8 μM for RPMI 8226, and 13 μM for U266. The decreased viability is attributed to cell-cycle arrest (U266, G1; RPMI 8226, G2M) and cell death. ARH77 underwent apoptosis, whereas U266 and RPMI 8226 displayed a more necrotic form of death. Cytoplasmic calcium levels decreased significantly in all treated cell lines. IL-6 secretion from U266 cells was abrogated on treatment with simvastatin, whereas total tyrosine phosphorylation was unaffected.ConclusionsSimvastatin displays significant antimyeloma activity in vitro. Further research is warranted for elucidation of the modulated molecular pathways and clinical relevance.

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