The Roles of HSP27 and Annexin II in Resistance to UVC-Induced Cell Death: Comparative Studies of the Human UVC-Sensitive and -Resistant Cell Lines RSa and APr-1

2009 ◽  
Vol 73 (6) ◽  
pp. 1318-1322 ◽  
Author(s):  
Yuan-Hu JIN ◽  
Kazuko KITA ◽  
Zhuo SUN ◽  
Xiao-Bo TONG ◽  
Hua NIE ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Lines ◽  
Comparative Studies ◽  
Resistant Cell ◽  
Annexin Ii

scholarly journals Elucidation of the IMiDs-Resistant Mechanism and Development of the Overcoming Drugs Inducing CRBN Independent G2/M Cell Cycle Arrest in Myeloma Cells

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4451-4451
Author(s):  
Ryo Uozaki ◽  
Shuji Aida ◽  
Takasi Yamagughi ◽  
Tomofumi Yamamoto ◽  
Sho Kashiwazaki ◽  
...  
Keyword(s):  
Cell Death ◽  
High Risk ◽  
Molecular Mechanism ◽  
Cell Lines ◽  
Research Funding ◽  
Water Solubility ◽  
Xenograft Model ◽  
Genetic Mutation ◽  
Resistant Cell ◽  
Tubulin Polymerization

Abstract [Introduction] Recent development of novel drugs significantly improved prognosis of Multiple Myeloma (MM). Immunomodulatory drugs (IMiDs) and proteasome inhibitors play central roles in MM therapy. However expanded usage of lenalidomide (Len) has increased the number of Len-resistant patients. And limited information is obtainable with regand to Len-resistant mechanism, such as overexpression and genetic mutation of IMiDs-binding protein, celeblon (CRBN); thus, elucidating the Len-resistant mechanism and development of drugs overcoming the Len-resistance are very important for improving the outcome of MM. The purposes of this study are [1] to clarify the molecular mechanism of Len-resistance using Len-resistant MM cell lines [2] to confirm the mechanism of CRBN independent myeloma cell death by novel phthalimide-derivatives, TC11 and PEG(E)-TC11. [Method] [1] In our laboratory, Len-resistant cell lines, KMS21R, KMS27R and MUM24R have been established by long-term co-culture with low-dose Len. Using these cell lines, we examined expression of CRBN and the downstream molecules, IKZF1/3, IRF4 and c-MYC by western blotting. We also examined the mutation of CRBN in KMS27R. [2] We have originally developed a novel phthalimide-derivative, TC11 and PEG(E)-TC11 synthesized for improving water solubility. We examined whether TC11 and PEG(E)-TC11 induced cell death to Len-resistance MM or not. [Result] [1] First, we validated expression of CRBN and the down-stream molecules, which mediate pharmacological action of Len. Decreased expression of CRBN and subsequent up-regulation of down-stream IKZF1 were confirmed in KMS21R cell. In KMS27R cell, IKZF 1/3 expressions are increased without alteration of CRBN expression level. Thus, genetic mutation in CRBN or IKZF1 is suspected in KMS27R cells. In MUM24R cell, no significant change in the expression levels of the CRBN pathway molecules was confirmed, suggesting other molecular alternation than CRBN pathway. [2] TC11 significantly induced apoptosis of Len-resistant cells. We have previously reported that TC11 didn't bind to CRBN and TC11 directly bound to nucleophosmin1 (NPM1) and α-tubulin. It was found that TC11 induced G2/M arrest and subsequent apoptosis by inhibition of tubulin polymerization and NPM1 oligomerization. Fluorescence microscopy observation showed that TC11 treatment induced hyper duplication of centrosomes in MM cells. Water solubility and blood absorption of PEG(E)-TC11 were significantly improved compared with those of TC11. As a consequence, PEG-(E)TC11 significantly delayed tumor growth in xenograft model mice. [Discussion & Conclusion] [1] Our present data suggested diversity of Len-resistant mechanism in MM patients. For example, in KMS21R, decreased expression of CRBN was likely the cause of Len-resistance. In KMS27R, genetic mutation in CRBN-IKZF1 pathway caused inhibition of IKZF1 degradation. In MUM24R, the Len-resistant mechanism didn't relate to the CRBN pathway but to unknown molecular mechanism. Len-resistant cell lines are useful tools for studying Len-resistant mechanisms and developing drugs overcoming Len-resistance. [2]TC11 abrogated tubulin polymerization and NPM1 oligomerization, induced centrosome disruption and G2/M arrest. Since G2/M check point doesn't closely rely on p53. TC11 was able to induce apoptosis of MM cells with high-risk cytogenetic mutations such as deletion of TP53 gene. TC11 and PEG-(E)TC11 are expected as a candidate compound overcoming Len-resistance and high-risk MM. Disclosures Matsushita: Amgen: Research Funding. Hattori:Takeda: Research Funding; IDAC inc.: Research Funding.

In Vitro Evaluation of Apoptosis and Cell Death of Neuroblastoma Cell Lines Exposed to Busulfan.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4459-4459
Author(s):  
Morris Kletzel ◽  
Sarah C. Tallman ◽  
Marie Olszewski ◽  
Wei Huang
Keyword(s):  
Cell Death ◽  
Cell Viability ◽  
Cell Lines ◽  
Neuroblastoma Cell ◽  
Annexin V ◽  
Resistant Cell ◽  
Primary Tumors ◽  
Induced Apoptosis ◽  
Neuroblastoma Cell Lines

Abstract Objective: While busulfan is a commonly used chemotherapeutic agent in the treatment of many hematological diseases, its effectiveness against neuroblastoma is still in question. This study aims to assess the degree of apoptosis and cell death in neuroblastoma cell lines and primary neuroblastoma tumors when exposed to varying doses of busulfan. Materials and Methods: Cultures from established cell lines SKN-SH, SKN-DOX-R, IMR-5, and NGP (n=4), as well as cultures from primary tumors (n=2) were seeded at 106 cells/ml in RPMI640 supplemented with 10% fetal bovine serum (FBS) and transferred to 24-well plates, where cells were exposed to 1ml of busulfan at 0, 0.001, 0.005, 0.01, 0.05, and 0.1mg/ml per well. Cells were incubated at 37°C in a humidified atmosphere of 5% CO2 for 72 hours. Wells were sacrificed after 0, 6, 24, 48 and 72 hours and tested with Annexin V and PI; 10,000 events were measured by flow cytometry. The percentage of apoptotic and dead cells was plotted in a graph and a t-test was performed against the untreated control. Results: After 24 hours, there was a significant decrease in cell viability of each dose when compared to the control untreated cells (p<0.005). 24 Hour % Cell Viability for Varying Doses of Busulfan (mg/ml) Dose 0 Dose 0.001 Dose 0.005 Dose 0.01 Dose 0.05 Dose 0.1 Mean 66.1 44.4 40.3 40.7 37.7 39 SEM 5.56 5.17 5.96 6.17 6.03 5.60 Median 65 33.5 38 39 37 31 Range 39 to 97 14 to 87 4 to 89 6 to 93 4 to 77 5 to 88 The overall mean decrease in cell viability when compared to the control was 25.7%. However, there were only modest differences in effectiveness when comparing the doses, with an average of only 5–7% difference between doses. Further, there was much variability between the different cell lines, some with changes in apoptosis and cell death of over 50%, while other lines showed no changes at all. Limited differences were seen after 6 hours, and after 72 hours any effect of busulfan was masked by cell death due to other factors, as seen through increased cell death in untreated cells. Conclusion: Busulfan induced apoptosis and cell death in vitro in neuroblastoma cell lines at a mean of 76.43% for non-resistant lines, 59.33% for primary tumors and 35% for resistant cell lines (at middle dose 0.01mg/ml). The resistance of certain cell lines confirms the difficulties of treating multi-drug resistant cells in often heterogeneous neuroblastoma tumors. That some cell lines were responsive shows the potential of using busulfan to treat neuroblastoma in the future.

Displacement of Bim From Anti-Apoptotic Proteins Is the Primary Factor for Determining ABT-737 Activity in Multiple Myeloma Cell Lines.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2851-2851
Author(s):  
Alejo A Morales ◽  
Metin Kurtoglu ◽  
David Siefker ◽  
Shannon M Matulis ◽  
Delia M Gutman ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Cell Lines ◽  
Cell Types ◽  
Myeloma Cell ◽  
Resistant Cell ◽  
The Other ◽  
Multiple Myeloma Cell ◽  
Other Hand

Abstract Abstract 2851 Poster Board II-827 ABT-737 and its orally active analog ABT-263 are Bcl-2-family inhibitors that are currently in clinical trials for a variety of cancers including hematological malignancies such as multiple myeloma. Previously, we reported that the sensitivity of multiple myeloma cell lines to ABT-737 correlates with the interactions, but not the expression, of Bcl-2 proteins. Analysis of 6 multiple myeloma cell lines revealed that expression of Bcl-2 proteins did not correlate with sensitivity, however the sensitive cells (8226/S, MM.1S and KMS-11) have a substantial amount of their pro-apoptotic Bcl-2 protein, Bak, bound to Bcl-xL. On the other hand, in the insensitive cell lines (U266, KMS-11 and OPM2), Bak was found to be associated with Mcl-1, a family member that does not bind ABT-737 and thereby confers resistance to this drug. Furthermore, we also showed that release of the BH3-only protein Bim by ABT-737 from Bcl-xL and Bcl-2 also contributes to cell death in 8226/S and MM.1S. The purpose of the current study is to further investigate the role of Bim in ABT-737-induced cell death in the multiple myeloma lines. Similar to Bak, a substantial amount of Bim is bound to Bcl-xL and Bcl-2 in the ABT-737-sensitive cell lines, MM.1S and KMS-18, while in the insensitive cell lines, it is highly bound to Mcl-1. Surprisingly, in the ABT-737-sensitive 8226/S cells, Bim appears to bind to Mcl-1. However in these cells, ABT-737 treatment resulted in upregulation of Noxa, which is a BH3-only protein that binds Mcl-1 and can release Bim. Taken together these data suggest that although binding of Bim to Mcl-1 may confer resistance to ABT-737, in certain cell types this treatment could also induce Noxa expression that antagonizes Mcl-1-mediated resistance. Consistent with this hypothesis, Mcl-1 overexpression as well as knockdown of Noxa expression significantly protected 8226/S cells from ABT-737-induced cell death while they had no effect in MM.1S cells. To further demonstrate the role of Bim in ABT-737-induced cell death, ABT-resistant 8226/S, KMS-11, KMS-18 and U266 cell lines were generated. In the resistant cell lines of 8226/S and KMS-18, Bim is exclusively bound to Mcl-1, which was overexpressed as compared to the parental cells. Bak binding was not affected by acquisition of ABT-737 resistance. This result is in agreement with the findings that interaction of Bim and Mcl-1 confers resistance to ABT-737. On the other hand, in ABT-resistant U266 and KMS-11 cell lines, Bim expression was down-regulated while Mcl-1 levels were not changed. Thus, it appears that in cells where Bim is already bound to Mcl-1, further resistance is achieved by down-regulating the expression of this BH3-only protein. Overall, these results suggest that the complex interactions between Bcl-2 proteins need to be investigated in order to understand how multiple myeloma cells may respond to ABT-737 treatment. Disclosures: Boise: University of Chicago: Patents & Royalties.

Mechanisms of Sensitivity and Resistance to Histone Deacetylase Inhibitors in Diffuse Large B-Cell Lymphoma

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1359-1359
Author(s):  
Ana A Tula-Sanchez ◽  
Aaron Havas ◽  
Peter Alonge ◽  
Mary E Klein ◽  
Taralyn Y Rogers ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Death ◽  
B Cell ◽  
Cell Cycle Arrest ◽  
Cell Lines ◽  
Histone Deacetylase Inhibitors ◽  
B Cell Lymphoma ◽  
Resistant Cell ◽  
G1 Arrest ◽  
Cycle Arrest

Abstract Abstract 1359 Diffuse large B-cell lymphoma (DLBCL) is the most common type of Non-Hodgkin Lymphoma (NHL) throughout the world. DLBCL is an aggressive, heterogeneous disease with two major recognized cell-of-origin subtypes: “germinal center” (GCB) and “activated B-cell like” (ABC), the latter having the worse prognosis. Overall, DLBCL remains fatal for about 30% patients due to relapse or lack of response to initial therapy. Resistant/relapsed DLBCL patients could benefit from the addition of new promising antiproliferative drugs, such as histone deacetylase inhibitors (HDACIs), to current chemotherapy regimens. So far, Vorinostat and Romidepsin, two structurally different HDACIs, have been approved for the treatment of hematological cancers. Despite their proven antiproliferative, pro-apoptotic effects, response to these drugs against DLBCL in clinical trials have been variable, ranging from complete/partial responses to stable disease to no response. The mechanisms of action of these drugs are still poorly understood, mainly because the function of their target deacetylases are cell context-specific. Therefore, characterization of the specific anticancer mechanisms of action of HDACIs in DLBCL could potentially lead to development of novel combinatorial drug regimens effective against resistant/relapsed DLBCL patients. To define HDACI action in DLBCL, we treated DLBCL-derived cell lines with PXD101, (Belinostat); a hydroxamate HDACI, like Vorinostat. We demonstrated that PXD101 is able to produce 24h growth inhibition (IC50) at submicromolar concentrations regardless of the DLBCL subtype. The 24h IC50values were used in all the subsequent experiments. Cell cycle and apoptosis analysis by flow cytometry indicated that PXD101 produces cytotoxic effects on two of the GCB cell lines; DB and OCILY19 underwent G2/M cell cycle arrest at 24 hours followed by apoptosis at 48 and 72 hours of treatment. Immunoblotting of PARP and caspase-3 cleavage further confirmed apoptosis. More importantly, when cells were treated for only 8 hours with PXD101 and then the drug was removed for 24 hours, cells showed apoptosis rates similar to those observed with 48h of continuous treatment; suggesting that once that these cell lines are exposed to the drug they rapidly commit to cell death. Thus, we have classified the DB and OCILY19 cell lines as models for sensitivity to the apoptotic effects of HDACI. In contrast, PXD101 induced cytostatic effects on the GCB cell line SUDHL4 and ABC cell lines U2932 and SUDHL8. All three cell lines showed G1 phase cell cycle arrest with little apoptosis. The G1 arrest is reversible after 48 hours of drug removal. Because of the lack of cell death and the reversibility of cell cycle arrest, we have classified these cell lines as models of HDACI resistance. Previous studies have shown that induction of p21 is responsible for G1 arrest in cells treated with HDACIs. Western blot analysis showed that none of the cell lines, except U2932, express p21, but upon PXD101, p21 protein levels were induced at 24, 48 and 72 hours of PXD101 treatment in SUDHL4 and U2932. In contrast, p21 was induced to a lesser extent in OCILY19 and DB, but its expression was not sustained beyond 24 hours of treatment. Since we also observed a corresponding loss in Rb phosphorylation, we tested the effect of PXD101 on cyclin dependent kinase 2 (CDK2) activity. This enzyme complex is responsible for entry into S phase and is inhibited by association with p21. In all three resistant cell lines CDK2 activity was reduced after only 24 hours of treatment with PXD101. The loss in activity was correlated with increased association with p21, as determined by immunoprecipitation. These results indicate that sustained upregulation of p21 by HDACIs such as PXD101 plays a role in bringing about G1 arrest that may protect DLBCL cells from apoptosis. Combined treatment with therapeutics that prevent p21 upregulation and G1 arrest may work synergistically with HDACIs to trigger apoptosis in HDACI-resistant cell lines. To that end, we have begun analysis of the cyclin-dependent kinase inhibitor, flavopiridol, and have shown that it prevents both p21 upregulation and G1 arrest in the HDACi-resistant DLBCL cell lines. Studies to measure synergism with PXD101 in bringing about cell death are currently underway. Disclosures: No relevant conflicts of interest to declare.

Mechanisms of Acquired Resistance to Venetoclax in Preclinical AML Models

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 328-328 ◽  
Author(s):  
Qi Zhang ◽  
Rongqing Pan ◽  
Lina Han ◽  
Ce Shi ◽  
Stephen E. Kurtz ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Lines ◽  
Small Molecule ◽  
Research Funding ◽  
Synergistic Effects ◽  
Acquired Resistance ◽  
Parental Cell ◽  
Resistant Cell ◽  
Flt3 Inhibitors ◽  
Resistant Cells

Abstract BH3-mimetic ABT-199 (venetoclax, VEN) is a selective small-molecule antagonist of the anti-apoptotic BCL-2 protein. It binds to BCL-2 specifically, causing the release of pro-apoptotic BAX and BH3-only proteins and induction of cell death. Our studies indicated that AML is a BCL-2 dependent disease that, in pre-clinical studies, responds robustly to VEN by induction of apoptotic cell death (Pan et al., Cancer Discovery 2014). As a single agent, VEN demonstrated clinical activity in relapsed/refractory AML, yet patients who initially responded ultimately developed resistance and progressed. In this study we investigated mechanisms of acquired resistance to VEN in preclinical AML models. First, we generated 5 VEN-resistant cell lines (OCI-AML2, Kasumi, KG-1, MV4;11 and Molm13; with VEN cell-killing IC50s of 0.021µM, 0.046µM, 0.073µM, 0.020µM and 0.050µM, respectively) by exposing the cells to gradually increasing VEN concentrations. The IC50s of resistant cells are 15.2µM, 5.7µM, 31.6µM, 11.4µM and 15.4µM (124-723-fold greater than their parental counterparts). Protein analysis of resistant cells using immunoblotting demonstrated increased expression of MCL-1, a known resistance factor to VEN, in 4 resistant cell lines (OCI-AML2, KG-1, Mv4;11 and Molm13); and BCL-XL increase in MV4;11 and Molm13 resistant cells. To characterize the functional role of MCL-1 and BCL-XL in resistance to VEN, we co-treated parental and resistant cells with novel MCL-1 and BCL-XL- selective inhibitors (A-1210477 and A-1155463). The combination of VEN with A-1210477 or A-1155463 showed synergistic growth inhibition in all 5 parental cell lines (combination indices (CI) for A-1210477 were 0.15-0.62; CI for A-1155463 were 0.33-0.51, except >3 for KG-1). Notably, 4 out of 5 resistant cell lines (OCI-AML2, Kasumi, MV4;11, Molm13) became more sensitive to MCL-1 selective inhibitor A-1155463 but not to BCL-XL inhibitor A-1210477. However, no further effects were seen in resistant cells when combined with VEN. We next compared sensitivity of three paired parental and resistant cell lines (OCI-AML2, MV4;11 and Molm13) to a library of 130 specific small-molecule inhibitors (Tyner, et.al.. Cancer Res. 2013). Cells were co-treated with VEN and each specific inhibitor, and drug target scores were calculated based on the IC50 of measured effectiveness of panel drugs against the cells. The screening revealed modulation of sensitivity to mTOR, MEK, and FLT3 pathways in resistant cells (Fig.1C). To confirm these findings, we next co-treated AML cells with VEN and specific inhibitors of the mTOR pathway (rapamycin and AZD2014) or MEK pathway (CI1040) in all 5 paired parental and resistant cell lines; or with FLT3 inhibitors (quizartinib and sorafenib) in parental and resistant MV4;11 and Molm13, which harbor FLT3-ITD. The combination of VEN and AZD2014 achieved synergistic effects in all 5 parental cell lines (CI AZD2014: 0.08-0.94), and VEN/rapamycin were synergistic in 3 parental cell lines (CI rapamycin: 0.00-0.55, except 1.76 for KG-1 and 1.59 for Molm13). Combination of VEN with CI1040 achieved synergy in OCI-AML2, Kasumi, MV4;11 and Molm13 parental cell lines (CI: 0.14-0.61). Finally, VEN/FLT3 inhibitors achieved synergistic effects in MV4;11 and Molm13 parental cell lines (CI quizartinib: 0.66-0.69; CI sorafenib: 0.64-0.71). The resistant cell lines exhibited sensitivity to these inhibitors as single agents, and no synergistic effects were seen when combined with VEN. We have further induced in vivo resistance in two primary AML xenografts by treating NSG mice engrafted with 2nd passage AML cells with 100 mg/kg Q.D. VEN for 4 weeks followed by harvest of leukemic cells that repopulated the mouse after treatment discontinuation. While the proteomics, gene expression (RNAseq) and drug screening assays are in progress, preliminary immunoblotting studies demonstrated decreased expression of BCL-XL and BCL-2 (Fig.1B). In summary, we identified multiple mechanisms of acquired resistance to VEN, which ultimately modulate the balance between pro- and anti-apoptotic BCL-2 family members. Our studies indicate that upfront combination of VEN with selective inhibitors of MCL-1, or with inhibitors of specific signaling pathways, can synergistically induce apoptosis in AML cells and conceivably prevent emergence of VEN resistance. Disclosures Leverson: AbbVie: Employment, Equity Ownership. Tyner:Aptose Biosciences: Research Funding; Constellation Pharmaceuticals: Research Funding; Janssen Pharmaceuticals: Research Funding; Array Biopharma: Research Funding; Incyte: Research Funding. Konopleva:Novartis: Research Funding; AbbVie: Research Funding; Stemline: Research Funding; Calithera: Research Funding; Threshold: Research Funding.

scholarly journals Retinoic Acid Enhances Drug-Induced Cell Death in Anticancer Drug-Resistant Cell Lines

2002 ◽  
Vol 34 (3) ◽  
pp. 212-217
Author(s):  
Young Mi Whang ◽  
Yeul Hong Kim ◽  
Sang Won Shin ◽  
Byung Soo Kim ◽  
Jun Suk Kim ◽  
...  
Keyword(s):  
Cell Death ◽  
Retinoic Acid ◽  
Cell Lines ◽  
Anticancer Drug ◽  
Resistant Cell ◽  
Drug Resistant ◽  
Drug Induced ◽  
Drug Resistant Cell

Comparative Phosphoproteomics Study Between Obinutuzumab (GA101) Vs. Rituximab (RTX) Against RTX Sensitive/Resistant Burkitt Lymphoma (BL): Differentially Phosphorylated B Cell Receptor, Fc-Gamma Receptor, Phagocytosis and Natural Killer Cell-Mediated Cytotoxicity Signaling Pathway Proteins

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1550-1550
Author(s):  
Aradhana Tiwari ◽  
Delphine CM Rolland ◽  
Mona Elmacken ◽  
Janet Ayello ◽  
Carmella van de Ven ◽  
...  
Keyword(s):  
Cell Death ◽  
Natural Killer Cell ◽  
B Cell ◽  
Signaling Pathways ◽  
Cell Line ◽  
Cell Lines ◽  
Killer Cell ◽  
Resistant Cell ◽  
Resistant Cell Line ◽  
Bcr Signaling

Abstract Background: Burkitt Lymphoma (BL) is the most common form of NHL in children and adolescents and has an excellent prognosis (≥80% 5years, EFS, Cairo et al. Blood, 2007, Cairo et al. JCO, 2012). The prognosis has improved with the addition of targeted immunotherapy with rituximab (Goldman/Cairo et al, Leukemia, 2013, Goldman/Cairo et al. BJH, 2014). However, a subset of patients with chemoimmunotherapy-resistant disease has a dismal prognosis (≤ 10% 5 years, EFS) (Miles/Cairo et al. BJH, 2012). Deregulation of signaling pathways controlled by protein phosphorylation underlies the pathogenesis of B-cell lymphomas, however, the extent to which they contribute to rituximab resistance is largely unknown (Barth et al. BJH, 2013). Obinutuzumab (GA101), a novel glycoengineered type II CD20 Ab vs. RTX, a Type I CD20 Ab, mediates enhanced cell death & ADCC against diffuse B-cell lymphoma vs. RTX (Mössner et al. Blood, 2010), and was recently approved by FDA and EMA for first line treatment of CLL in combination with chlorambucil. Objective: To evaluate phosphorylation of signaling pathways are differentially altered following obinutuzumab vs RTX against RTX-sensitive/resistant BL. Methods: Raji (CD20+, ATCC, Manhass, VA) and Raji-4RH (provided by M. Barth, Roswell Park Cancer Institute) were cultured in RPMI with 10% FBS. For in-vitro studies, tumor cells were incubated with 100 µg/ml obinutuzumab (supplied by Christian Klein, PhD, Roche Research & Early Development, Zurich), and/or RTX for 24 hrs. For Phosphoproteomics analysis, we performed a mass spectrometry-based label-free quantitative phosphoproteomic profiling of the BL cell lines Raji/Raji4RH in the presence/absence of obinutuzumab or rituximab (100µg/ml for 24h) or isotype control. Six milligrams of protein from each condition were digested by trypsin and peptides and subjected to phosphopeptide enrichment using metal oxide affinity chromatography (MOAC) and immunoprecipitation. An LTQ Orbitrap XL, in-line with a Paradigm MS2 HPLC was employed for acquiring high-resolution MS and MS/MS data that were searched with the Swissprot Human taxonomic protein database (McDonnell and Lim et al, Blood, 2013). Silencing of PLCG2 in Raji and Raji4RH cell lines was carried out according to the manufacturer's instructions (Dharmacon, PA, USA). Results: Four hundred and eighteen out of total 661 proteins in Raji and 377 out of total 534 proteins in Raji4RH were differentially phosphorylated (>1.5 fold) after obinutuzumab treatment. Of these proteins, 46 were expressed at significantly higher levels in obinutuzumab vs. RTX in Raji (Figure 1). However, Raji4RH, the RTX resistant cell line did not show significant increase in phosphorylation of protein following in obinutuzumab vs. RTX. Proteins differentially phosphorylated in response to obinutuzumab vs. RTX are involved in the BCR (PLCG2, BTK, GSK3B and RAF-1), FC gamma phagocytosis (FCRG2B, MAPK1, PLCG2 and RAF-1), and Natural Killer cell-mediated cytotoxicity (MAPK1, RAF-1, PLCG2 and MAPK3) signaling pathways in Raji vs. Raji4RH (Figure 2). Differential phosphorylation of BCR signaling pathways proteins (BTK, PLCG2 and GSK3B), validated by western blot studies after incubation with obinutuzumab vs. RTX in Raji/Raji4RH cell lines, revealed up-regulation of BTK and PLCY2 after obinutuzumab treatment vs. RTX treatment in Raji BL cell line. Silencing one of the BCR signaling pathway protein, PLCG2 significantly increased cell proliferation and decreased cell death after obinutuzumab vs. RTX treatment in Raji (P=0.0001 & 0.004) however, there was no change in Raji 4RH RTX resistant cell line. Conclusions: Obinutuzumab and RTX differentially phosphorylate BCR, phagocytosis and cytotoxicity signaling pathways in BL. Knockdown of PLCG2 significantly enhanced BL proliferation and reduced cell death after obinutuzumab vs. RTX treatment. These results offer insights into alternate therapeutic strategies in the treatment of RTX resistant BL. Disclosures Klein: Roche: Employment.

scholarly journals 4-IPP, a selective MIF inhibitor, causes mitotic catastrophe in thyroid carcinomas

2015 ◽  
Vol 22 (5) ◽  
pp. 759-775 ◽  
Author(s):  
Luca Varinelli ◽  
Dario Caccia ◽  
Chiara C Volpi ◽  
Claudio Caccia ◽  
Maida De Bortoli ◽  
...  
Keyword(s):  
Cell Death ◽  
Thyroid Carcinoma ◽  
Cell Lines ◽  
Thyroid Neoplasms ◽  
Mitotic Cell ◽  
Resistant Cell ◽  
Anaplastic Thyroid Carcinoma ◽  
Mitotic Catastrophe ◽  
Important Player ◽  
Cell Subpopulations

Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine that is over-expressed in several human neoplastic cells. When MIF binds its receptor (CD74) and co-receptor (CD44), it initiates signaling cascades that orchestrate cell proliferation and survival, and it can directly modulate the activity of AMPK. These activities indicate that MIF potentially regulates cell survival and metabolism. We found that MIF was primarily co-expressed with CD74 in 16 out of 23 papillary thyroid carcinoma (PTC) and in all the 27 available anaplastic thyroid carcinoma (ATC) biopsy samples. MIF and CD74 were co-expressed in TPC-1 and HTC-C3 cell lines. The selective MIF inhibitor, 4-iodo-6-phenylpyrimidine (4-IPP), blocked MIF/CD74 internalization, activated JNK, and dose-dependently inhibited proliferation inducing apoptosis and mitotic cell death. In two CD74-negative cell lines, NIM-1 and K1, 4-IPP treatment partially reduced proliferation. Coordinated MIF and CD74 expression appeared to confer in tumor cells the plasticity necessary to escape cell cycle regulation, metabolic changes, and stress conditions. MIF/CD74 signaling removal made cells susceptible to apoptosis and mitotic cell death. This finding suggests a possible avenue for targeting DNA endoreduplication, thus preventing the proliferation of therapy-resistant cell subpopulations. This study highlights MIF/CD74 axis as an important player in the biology of aggressive thyroid neoplasms.

scholarly journals Upregulation of MAPK/MCL-1 Maintaining Mitochondrial Oxidative Phosphorylation Confers Acquired Resistance to BCL-2 Inhibitor Venetoclax in AML

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 101-101 ◽  
Author(s):  
Qi Zhang ◽  
Lina Han ◽  
Ce Shi ◽  
Rongqing Pan ◽  
Man Chun John MA ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Lines ◽  
Research Funding ◽  
Acquired Resistance ◽  
Resistant Cell ◽  
Mitochondrial Activity ◽  
Mitochondrial Oxidative Phosphorylation ◽  
Expression Of Genes ◽  
Apoptotic Proteins ◽  
Resistant Cells

Abstract ABT-199 (venetoclax), a selective small-molecule antagonist of the anti-apoptotic protein BCL-2, enables the activation of pro-apoptotic proteins and the induction of cancer cell death. Our previous studies found that AML is a BCL-2 dependent disease and responds robustly to venetoclax by induction of apoptotic cell death (Pan et al., Cancer Discovery 2014). Despite initial responses to single agent venetoclax in a Phase II trial of relapsed AML, patients ultimately developed resistance and progressed (Konopleva et al., Cancer Discovery 2016). In this study we investigated mechanisms of acquired resistance to venetoclax in preclinical AML models. First, we generated 5 AML cell lines resistant to ³1µM venetoclax. No BAX (exon5 and 6) or BCL2 (exon2) mutations were found in resistant cells. Immunoblotting analysis demonstrated increased expression of anti-apoptotic proteins MCL-1, BCL-2 A1, and BCL-XL, and a decrease of pro-apoptotic PUMA protein in selected resistant cell lines. To probe the functional interactions between the pro- and anti-apoptotic proteins, we next performed co-immunoprecipitation (co-IP) studies. The anti-BIM and anti-MCL-1 co-IPs revealed reduced levels of BIM:BCL-2 complexes and increased BIM:MCL-1 complexes in resistant cells compared to their parental counterparts (Fig 1B). The BH3 profiling technique examines mitochondrial sensitivity to different BH3 mimetic peptides, and has proven to be a useful tool to determine cell dependence on anti-apoptotic BCL-2 family proteins. BH3 profiling demonstrated that resistant cells had increased responses to NOXA, MS1 and HRK peptides, indicating increased dependence on MCL-1 and/or BCL-XL (Fig 1C). To characterize the functional role of MCL-1 in resistance to venetoclax, we co-treated parental and resistant cells with selective BCL-XL or MCL-1 inhibitors A-1155463 (Leverson et al. Science Transl Med 2015) and A-1210477 (Leverson et al., Cell Death Dis 2015). The combination of venetoclax with either A-1155463 or A-1210477 showed synergistic growth inhibition in all 5 parental cell lines. Notably, 4 of the 5 resistant cell lines (OCI-AML2, Kasumi, MV4-11, MOLM13) became more sensitive to an MCL-1 inhibitor but not to a BCL-XL inhibitor (Fig 1E). However, no further sensitization was seen in combination with venetoclax in resistant cells. To characterize additional mechanisms of resistance to venetoclax in AML cells, we conducted RNA sequencing of single cell clones (2 clones/cell line) isolated from paired isogenic cells (OCI-AML2, MV4-11, MOLM13). Analysis of RNA expression patterns by gene set enrichment analysis (GSEA) revealed elevated expression of genes in the RAS/MAPK pathway (Fig 1F), consistent with increased p-ERK and p-p90-RSK protein levels (Fig 1G). Inhibition of MAPK with MEK inhibitor GDC-0973 reduced MCL-1 expression in parental but not in resistant cells, indicating that MAPK activation partially contributed to high MCL-1 levels (Fig 1G). GSEA of RNAseq data further uncovered altered expression of genes involved in mitochondrial oxidative phosphorylation (OxPhos) in 3 resistant cell lines with high MCL-1 expression (OCI-AML2, MV4-11 and MOLM-13). Notably, BCL-2 was reported to sustain AML stem cell survival through maintenance of the mitochondrial activity of OxPhos (Lagadinou etal., Cell Stem Cell, 2013). Analysis of mitochondrial respiration using a Seahorse Bioanalyzer demonstrated similar levels of oxygen consumption rate (OCR) in parental and resistant cells. Inhibition of BCL-2 with 100nM venetoclax for only 2 hrs. fully blocked baseline and maximal respiratory activity in parental but not in resistant cells. In turn, inhibition of MCL-1 with A-1210477 inhibited respiration in both parental and resistant cells, indicating a role for MCL-1 in sustaining mitochondrial activity in venetoclax-resistant AML cells, which can maintain unperturbed mitochondrial function. In summary, we identified a novel mechanism of resistance to targeted BCL-2 inhibition through upregulation of MAPK leading to increased levels of anti-apoptotic MCL-1 that binds and neutralizes BIM and maintains the mitochondrial OxPhos pathway in AML cells. Concomitant inhibition of BCL-2 and MCL-1, or of BCL-2 and OxPhos could induce synergistic cell death in AML and conceivably prevent the emergence of venetoclax resistance. Disclosures Tyner: Constellation Pharmaceuticals: Research Funding; Janssen Research & Development: Research Funding; Agios Pharmaceuticals: Research Funding; Genentech: Research Funding; Array Biopharma: Research Funding; Inctye: Research Funding; Seattle Genetics: Research Funding; Aptose Biosciences: Research Funding; AstraZeneca: Research Funding; Takeda Pharmaceuticals: Research Funding; Leap Oncology: Consultancy. Leverson:AbbVie: Employment, Other: Shareholder in AbbVie. Letai:Astra-Zeneca: Consultancy, Research Funding; Tetralogic: Consultancy, Research Funding; AbbVie: Consultancy, Research Funding. Konopleva:Calithera: Research Funding; Cellectis: Research Funding.

The BH3-Mimetic Obatoclax (GX15-070) Posses a Dual-Mechanism of Action and Induces Both Apoptosis and Autophagy-Dependent Cell Death of B Cell Non-Hodgkin’s Lymphoma (B-NHL) Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 605-605 ◽  
Author(s):  
Francisco J. Hernandez-Ilizaliturri ◽  
Sapna Khubchandani ◽  
Scott H. Olejniczak ◽  
Paul Hosking ◽  
Elizabeth Gruber ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Cell Death ◽  
B Cell ◽  
Cell Lines ◽  
Mechanism Of Action ◽  
Resistant Cell ◽  
Dependent Cell ◽  
Dual Mechanism ◽  
Anti Tumor Activity ◽  
Bh3 Mimetic

Abstract The Bcl-2 family of proteins represents a rheostat that controls cellular viability. Aberrant expression of these proteins is often found in B-cell non-NHL and is known to affect treatment outcomes in B-cell lymphoma patients. Novel compounds such as the BH3-mimetic Obatoclax have been designed to specifically target and counteract anti-apoptotic Bcl-2 family proteins. We previously demonstrated that Obatoclax was able to enhance the anti-tumor activity of rituximab and chemotherapy agents. In the current work, we studied the mechanisms by which Obatoclax induces its anti-tumor effects. Studies were conducted in rituximab-chemotherapy sensitive and resistant cell lines and in malignant B-cells derived from patients with B cell non-Hodgkin’s lymphoma (n = 15). Cells were exposed to Obatoclax for 24 or 48 hrs with and without caspase inhibitors. Viability was evaluated using the cell-titer glow-assay. Induction of apoptosis was evaluated by flow cytometry and Western blotting. Autophagy was detected by LC3 conversion and electron microscopy. In addition, changes in p53, Noxa, Puma, and Bid following Obatoclax exposure were evaluated by Western blotting. Transient knock-down of p53 and beclin as well as pre-treatment of NHL cells to 3-methyladenine were performed to determine the role of of p53 or autophagy in the anti-tumor activity of Obatoclax, respectively. We found that Obatoclax induced time- and dose-dependent cell death of therapy-sensitive and –resistant cell lines along with all ex vivo treated patient samples tested to date (n = 15). In addition, in vitro exposure of NHL cells to Obatoclax resulted in significant downregulation of p53 and subsequent induction of Noxa and PUMA. Interestingly, the ability of Obatoclax to induce caspase-dependent PARP cleavage varied between patient samples and was not observed in therapy-resistant cell lines (TRCL). In Obatoclax rituximab-senstive treated cells, apoptosis was detected by APC-annexin staining. Inhibition of caspase activity by zVAD-fmk did not affect the ability of Obatoclax to kill B-NHL cell lines, suggesting that Obatoclax did not induce caspase-dependent apoptotic cell death of B-NHL cells. Induction of autophagy was detected by LC3 conversion not only in rituximab-chemotherapy sensitive or resistant cell lines but also in patient-derived tumor cells. Furthermore, electron microscopy confirmed the induction of autophagy following Obatoclax treatment of rituximab-chemotherapy resistant cells. Additionally, cell death induced by Obatoclax could be inhibited by knockdown of Beclin-1 or p53 and by treatment with 3-methyladenine. Together, these data suggest that Obatoclax has a dual mechanism of action and is capable to induce apoptosis or autophagy in B-cell NHL cells. We also provide data suggesting that both p53 and Beclin-1 have a pivotal role in response to Obatoclax and suggesting a crosstalk between p53, BH3 single domain proteins and autophagy. A better understanding in the molecular events triggered by Obatoclax and other Bcl-2 inhibitors is necessary in order to develop novel combination strategies using these exciting agents.

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