Establishment and Indentification of Genetic Alterations in Chemotherapy-Resistant T-ALL Cell Lines.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4423-4423
Author(s):  
David A. Estes ◽  
Rahul Poria ◽  
Debbie M. Lovato ◽  
Hadya M. Khawaja ◽  
Claudia L. Quan ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Cell Line ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Acquired Resistance ◽  
Genetic Alterations ◽  
Resistant Cell ◽  
Multi Drug Resistance ◽  
Growth Media ◽  
Drug Resistant

Abstract Acquisition of drug resistance in tumor cells in children with T-cell acute lymphoblastic leukemia (T-ALL) during chemotherapy results in relapse and poor outcome. T-ALL cell lines that have acquired resistance to chemotherapeutics are therefore critical tools for the study of acquired resistance, yet there is a paucity of cell lines available for study. In this study, we hypothesize that drug resistant T-ALL cells can be produced by prolonged exposure to chemotherapeutics and that microarray analysis can be employed to identify the gene products responsible for acquired drug resistance. By incrementally increasing the drug concentration in growth media, we have produced T-ALL cell lines (Jurkat and Sup T1) that grow well in the presence of therapeutic concentrations of L-asparaginase (ASNase) and daunorubicin (DNR). The genetic profiles of the drug-resistant cell lines were compared to their parental progenitors using the Affymetrix HG-U133Plus2 GeneChip platform, capable of hybridizing ~54,000 genes and ESTs/chip. Signal intensity was normalized using the robust multi-array average (RMA) technique in GeneSpring 7.2. The Sup T1 and Jurkat ASNase-resistant cell lines increased their IC50s 26-fold (0.044 IU/mL to 1.14 IU/mL) and 320-fold (0.003 IU/mL to 0.96 IU/mL), respectively. The IC50 of the Jurkat DNR resistant cell line increased 77-fold (30 nM to 2300 nM), and 4.0-fold, (0.46 nM to 1.85 nM), respectively. Notably, DNR resistant Jurkat cells were also resistant to therapeutic concentrations of vincristine and prednisolone, but not ASNase. In contrast, the ASNase resistant cell lines remained sensitive to DNR, vincristine, and prednisolone. Microarray data comparing DNR-resistant and parental cell lines showed 288 genes upregulated >1.5-fold in the resistant line. Two sets of genes were the most upregulated in the drug resistant cells in comparison to parental cells. ABCB1 (ABC transporter P-glycoprotein) was upregulated ~940-fold and genes coding for 6 different killer-cell immunoglobulin-like receptors (KIRs) were upregulated >6-fold. In the case of the ASNase-resistant cell lines, 96 genes were found to be upregulated >1.5-fold in both Jurkat and Sup T1 lines. The most highly upregulated gene in both cell lines was argininosuccinate synthase (ASS), 32-fold upregulated in Jurkat and 6.5-fold in Sup T1. All expression results were confirmed by qRT-PCR. These genes have previously been implicated in the acquisition of drug resistance: ASS is critical for responding to asparagine depletion caused by ASNase. ABCB1 acts as a molecular pump capable of lowering intracellular concentrations of substrate chemotherapeutics such as DNR, vincristine, and prednisolone, consistent with our observation of multi-drug resistance in that cell line. To our knowledge, this is the first description of DNR and ASNase resistant Jurkat and Sup T1 T-ALL cell lines. In addition, our results suggest that microarray technology is a valid method for elucidating the genetic nature of drug resistance in T-ALL cell lines, making it a productive approach to identify mechanisms of chemotherapy resistance. Finally, these cell lines will serve as useful tools for studying mechanisms of chemotherapeutic resistance in T-ALL.

Resistance to the Novel Translation Inhibitor Silvestrol Is Mediated by Elevated Mcl-1 Expression.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1737-1737
Author(s):  
David M. Lucas ◽  
Ellen J. Sass ◽  
Ryan B. Edwards ◽  
Li Pan ◽  
Gerard Lozanski ◽  
...  
Keyword(s):  
Drug Resistance ◽  
T Cells ◽  
B Cells ◽  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Multi Drug Resistance ◽  
Parental Line

Abstract Abstract 1737 Poster Board I-763 We previously reported the efficacy and B-cell selectivity of the natural product silvestrol in acute lymphoblastic leukemia (ALL) and chronic lymphocytic leukemia (CLL), using both primary cells and B-cell lines. We also showed that silvestrol inhibits translation, resulting in rapid depletion of the short half-life protein Mcl-1 followed by mitochondrial damage and apoptosis. Cencic et al. reported that silvestrol directly blocks translation initiation by aberrantly promoting interaction of eIF4A with capped mRNA (PLoS One 2009; 4(4):e5223). However, the loss of Mcl-1 in breast and prostate cancer cell lines is delayed relative to what we observe in B-leukemias (48 hr vs. 4-6 hr in CLL and ALL cells). Additionally, silvestrol does not reduce Mcl-1 expression in normal T-cells to the same extent that it does in B-cells, potentially explaining in part the relative resistance of T-cells to this agent. We therefore investigated cell-type differences, as well as the importance of Mcl-1, in silvestrol-mediated cytotoxicity. We incubated the ALL cell line 697 with gradually increasing concentrations of silvestrol to generate a cell line (697-R) with resistance to 30 nM silvestrol (IC50 of parental 697 < 5 nM). No differences between 697-R and the parental line were detected upon detailed immunophenotyping. However, cytogenetic analysis revealed a balanced 7q;9p translocation in 697-R not present in the parental 697 cell line that may be related to the emergence of a resistant clone. We also detected no difference in expression of multi-drug resistance proteins MDR-1 and MRP, which can contribute to resistance to complex amphipathic molecules such as silvestrol. In contrast, we found that baseline Mcl-1 protein expression is strikingly increased in 697-R cells relative to the parental line, although these cells still show similar percent-wise reduction in Mcl-1 upon re-exposure to 80 nM silvestrol. To investigate whether this resistance to silvestrol is reversible, 697-R cells were maintained without silvestrol for 6 weeks (∼18 passages). During this time, viability remained near 99%. Cells were then treated with 30 nM silvestrol. Viability was 94% at 48 hr post-treatment and returned to 99% within a week, while parental 697 cells with the same treatment were completely dead. Baseline Mcl-1 levels remained elevated in 697-R even with prolonged silvestrol-free incubation. These results indicate that the resistance phenotype is not rapidly reversible, as is seen with transient upregulation of multi-drug resistance or stress-response proteins. Additionally, silvestrol moderately induces the transcription of several pro-apoptotic Bcl-2 family members and results in elevated levels of these proteins despite its translation inhibitory activity. Interestingly, no such activity is detected in silvestrol-treated normal T-cells. Together, these results support the hypothesis that in B-cells, silvestrol induces cell death by altering the balance of pro- and anti-apoptotic factors, and that increased Mcl-1 protein can force the balance back toward survival. This work further underscores the importance of Mcl-1 in silvestrol-mediated cytotoxicity. We are now investigating the mechanism of Mcl-1 upregulation in 697-R cells to identify a factor or pathway that can be targeted therapeutically to circumvent resistance. Silvestrol is currently undergoing preclinical pharmacology and toxicology investigation by the U.S. National Cancer Institute Drug Development Group at the Stage IIA level to facilitate its progression to Phase I clinical testing. Disclosures No relevant conflicts of interest to declare.

scholarly journals Understanding the Role of Extracellular Vesicles in Lenalidomide-Resistance Multiple Myeloma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1887-1887
Author(s):  
Tomofumi Yamamoto ◽  
Nobuyoshi Kosaka ◽  
Ochiya Takahiro ◽  
Yutaka Hattori
Keyword(s):  
Bone Marrow ◽  
Drug Resistance ◽  
Cell Line ◽  
Cell Lines ◽  
Parental Cell ◽  
Resistant Cell ◽  
Bone Marrow Microenvironment ◽  
Drug Resistant ◽  
Apoptosis Assay ◽  
Drug Resistant Cell

Abstract Multiple myeloma (MM) is a plasma cell malignancy that develops by the accumulation of various genetic abnormalities. In recent years, the prognosis of MM has improved by the development of novel drugs including immunomodulatory drugs (IMiDs), proteasome inhibitors, and antibody drugs. However, MM cells acquired drug resistance by long-term exposure to these therapeutic drugs. MM is a multiclonal disease, and various clone subtypes develop within the bone marrow microenvironment. It has been suggested that drug resistant phenotype could transmit from resistant clones to sensitive ones, and also to immune cells, or mesenchymal stem cells, resulting in the change of bone marrow microenvironment suitable for MM cell survival. It has been shown that extracellular vesicles (EVs) are one of the means of information transmission. EVs are secreted from almost all cells, and the amount of EV secretion is particular high from cancer cells. It was already known that cancer-derived EVs transmitted information associated with cancer progressions such as angiogenesis, metastasis, and drug resistance to the surrounding cells. Thus, EVs were proposed to play an important role in acquisition of drug resistance even though the mechanisms have not been fully understood in MM. In order to understand the mechanism of drug resistance in MM mediated by EVs, lenalidomide resistant cell lines were established by long-term exposure of lenalidomide. The amount of EVs was measured by ExoScreen, which is ultra-sensitive detection method of EVs by measuring surface protein of EVs, such as, CD9 and CD63, and by the nanoparticle tracking analysis. We found that lenalidomide resistant cell lines in KMS21R, KMS27R and KMS34R cell lines secreted about twice more EVs than their parental cell lines, and the amount of EV secretion was correlated with the drug sensitivity of lenalidomide. Suppression of EV secretion by knockdown of TSG101, which is known for EV secretion-associated protein, did not affect lenalidomide resistance. We could suppress the EV secretion to two-thirds, however cell proliferation and caspase activity were not change. From these results, we postulated the two possibilities; 1) EV secretion pathway other than TSG101 is associated with drug resistance via EVs; 2) EV derived from lenalidomide resistant cells can affect the cells exist in bone marrow microenvironment. From these hypotheses, we have done the following experiments. Firstly, to identify the genes which involved in EV secretion pathway associated with drug resistance, RNA sequence among the drug-resistant cell lines and their parental cell lines was performed. Drug resistant cell lines had some genetic abnormality, for instance immune system or angiogenesis. Now, we are detecting the EV secretion associated genes in drug resistant cell lines. Secondly, EV derived from the drug-resistant cell lines and EV derived from the parent cell lines were added to drug sensitive MM cell lines, then lenalidomide is added after 24hr. The cell proliferation and apoptosis assay were evaluated after 48hr. EV derived from the drug-resistant cell lines in KMS34R cell line significantly inhibited cell death measured by MTS assay and apoptosis assay compared with those from the drug sensitive KMS21 and KMS34 cell lines. EVs from KMS34R cell line, which is the most progressed cell line we established, could more transmit drug resistance than those from other cell lines. These results suggested that drug resistance was transmitted from drug-resistant cell lines to non-resistant cell lines via EVs. Now, we are analyzing the component of EV from drug-resistant MM cells by proteome analysis to identify the molecules associated with lenalidomide resistance in MM. In addition, we are investigating the molecules which associated with the secretion of EVs from drug-resistance MM. These results prompted us to hypothesize that attenuating the function of a molecule responsible for EV secretion could lead to the inhibition of cancer development such as drug resistance. It is expected that EVs will be novel therapeutic targets in refractory or relapsed MM. Disclosures Hattori: IDAC inc.: Research Funding; Takeda: Research Funding.

scholarly journals Proteometabolomics of Melphalan Resistance in Multiple Myeloma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5619-5619
Author(s):  
David C. Koomen ◽  
Joy D. Guingab ◽  
Paula S. Oliveira ◽  
Bin Fang ◽  
Min Liu ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Cell Line ◽  
Cell Lines ◽  
Metabolic Pathways ◽  
Acute Treatment ◽  
Alkylating Agents ◽  
Resistant Cell ◽  
Untargeted Metabolomics ◽  
Drug Resistant ◽  
Endogenous Metabolites

Abstract Although advancements in therapeutic regimens for treating multiple myeloma (MM) have prolonged patient survival, the disease remains incurable. Several classes of drugs have contributed to these improvements, such as proteasome inhibitors, immunomodulators, deacetylase inhibitors, monoclonal antibodies, and alkylating agents including melphalan. An expanded arsenal of diverse chemotherapy targets has improved patient care significantly, yet we still lack sufficient knowledge of how cellular metabolism and drug processing can contribute to drug resistance. To address this issue, we utilize cell line models to simulate naïve and drug resistant states, which identify drug modifications, endogenous metabolites, proteins, and acute metabolic profile alterations associated with therapeutic escape. Here, we specifically focus on melphalan; an alkylating agent that forms DNA interstrand crosslinks, inhibits cell division, and leads to cell death through apoptosis (Povirk & Shuker. Mutat. Res. 1994, 318, 205). Melphalan remains a critical component of high dose therapy in the context of stem cell transplant and induction therapy in transplant ineligible patients outside the US. Ineffectiveness of alkylating agents remains a critical problem and serves as an excellent model for investigation of cellular metabolism and its contribution to drug resistance. Two parental MM cell lines (8226 & U266) were obtained from ATCC and resistant derivatives of each cell line (8226-LR5 & U266-LR6) were selected after chronic drug exposure. To assess mechanisms of melphalan resistance, we use liquid chromatography-mass spectrometry-based metabolomics and proteomics approaches, including studies of drug metabolism, untargeted metabolomics, and activity based protein profiling (ABPP). Drug metabolism monitors the intracellular and extracellular drug modifications over a 24-hour period after acute treatment. Untargeted metabolomics is used to compare the steady state endogenous intracellular metabolites of naïve and drug resistant cells. Differences in endogenous metabolites between naïve and drug resistant cell lines are also examined in the acute treatment dataset. ABPP utilizes desthiobiotinylating probes to enrich for ATP-utilizing enzymes, which are identified and quantified to enable comparison. We initially compared acute melphalan treatment in drug naive and resistant isogenic cell line pairs. Predictably, melphalan was converted into monohydroxylated and dihydroxylated metabolites more quickly in cells than in media controls. Differences in the formation of these metabolites between the naïve and resistant cell lines were not observed. The untargeted metabolomics data indicated in the 8226-LR5 model, glutathione and xanthine levels are elevated, while guanine is suppressed relative to naive cells. ABPP demonstrated changes in several enzymes related to purine and glutathione metabolism (Figure 1). Interestingly, the U266/U266-LR6 cell line models exhibit higher baseline levels of glutathione when compared with 8226/8226-LR5, indicating heterogeneous means of drug resistance. Alterations in arginine biosynthesis and nicotinate/nicotinamide metabolism are observed in the untargeted metabolomics and ABPP of U266/U266-LR6. Common pathways (e.g. purine biosynthesis) are altered in both models, although the changes involve different molecules. In examining two models of acquired melphalan resistance, we demonstrate frank differences in metabolic pathways associated with steady state and acute drug response. These data demonstrate the potential heterogeneity in drug resistance mechanisms and the need for more biomarkers to personalize treatment. Ongoing studies involve introduction of enzyme inhibitors in targeted pathways and supplementation of metabolites to validate their role in resistance. Furthermore, we will examine expression of these metabolic pathways associated with ex vivo melphalan resistance in a cohort of over 100 patient samples with paired RNA sequencing. The long term goals are to elucidate mechanisms of therapeutic response, identify biomarkers of metabolism in melphalan resistance, enhance drug efficacy, predict personalized patient treatment, and improve overall MM patient care. Disclosures No relevant conflicts of interest to declare.

scholarly journals Dye-mediated photolysis is capable of eliminating drug-resistant (MDR+) tumor cells

Blood ◽  
1993 ◽  
Vol 81 (3) ◽  
pp. 793-800 ◽  
Author(s):  
RM Lemoli ◽  
T Igarashi ◽  
M Knizewski ◽  
L Acaba ◽  
A Richter ◽  
...  
Keyword(s):  
Cell Line ◽  
Tumor Cells ◽  
Cell Lines ◽  
Light Exposure ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cell ◽  
Myelogenous Leukemia ◽  
Leukemia Cell Line ◽  
Drug Resistant ◽  
Colony Forming Unit

Abstract We evaluated the potential role of photoradiation therapy with a benzoporphyrin derivative, monoacid ring A (BPD-MA), and dihematoporphyrin ether (DHE), for the ex vivo purging of residual tumor cells from autologous bone marrow (BM) grafts. BPD-MA and DHE photosensitizing activity was tested against two human large-cell lymphoma cell lines and colony-forming unit-leukemia (CFU-L) derived from patients with acute myelogenous leukemia (AML). In mixing experiments, 4-log elimination of tumor cell lines was observed after 1 hour of incubation with 75 ng/mL of BPD-MA or 30 minutes of treatment with 12.5 micrograms/mL of DHE followed by white light exposure. By comparison, using the same concentration of BPD-MA, the mean recovery of normal BM progenitors was 4% +/- 0.8% (mean +/- SD) for granulocyte- macrophage colony-forming unit (CFU-GM) and 5% +/- 0.8% for burst- forming unit-erythroid (BFU-E). Similarly, DHE treatment resulted in the recovery of 5.2% +/- 2% and 9.8% +/- 3% of CFU-GM and BFU-E, respectively. Furthermore, equivalently cytotoxic concentrations of both DHE and BPD-MA and light were found not to kill normal pluripotent stem cells in BM, as demonstrated by their survival in two-step long- term marrow culture at levels equal to untreated controls. The T- lymphoblastic leukemia cell line CEM and its vinblastine (VBL)- resistant subline CEM/VBL, along with the acute promyelocyte leukemia cell line HL-60 and its vincristine (VCR)-resistant subline HL-60/VCR, were also tested. BPD-MA at 75 ng/mL was able to provide a greater than 4-log elimination of the drug-sensitive cell lines, but only a 34% and 55% decrease of the drug-resistant HL-60/VCR and CEM/VBL cell lines, respectively. On the contrary, 12.5 micrograms/mL of DHE reduced the clonogenic growth of all the cell lines by more than 4 logs. Further experiments demonstrated decreased uptake of both BPD-MA and DHE by the resistant cell lines. However, all the cell lines took up more DHE than BPD-MA under similar experimental conditions. Our results demonstrate the preferential cytotoxicity of BPD-MA and DHE toward neoplastic cell lines and CFU-L from AML patients. In addition, DHE was slightly more effective in purging tumor cells expressing the p-170 glycoprotein. These results suggest that photoradiation with DHE would be useful for in vitro purging of residual drug-resistant leukemia and lymphoma cells.

scholarly journals Expression of Osteoblast-Specific Factor 2 (OSF-2, Periostin) Is Associated with Drug Resistance in Ovarian Cancer Cell Lines

2019 ◽  
Vol 20 (16) ◽  
pp. 3927 ◽  
Author(s):  
Karolina Sterzyńska ◽  
Dominika Kaźmierczak ◽  
Andrzej Klejewski ◽  
Monika Świerczewska ◽  
Karolina Wojtowicz ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Drug Resistance ◽  
Cell Line ◽  
Cell Lines ◽  
Cancer Cell ◽  
Ovarian Cancer Cell ◽  
Ovarian Cancer Cell Line ◽  
Cancer Cell Line ◽  
Specific Factor ◽  
Drug Resistant

One of the main obstacles to the effective treatment of ovarian cancer patients continues to be the drug resistance of cancer cells. Osteoblast-Specific Factor 2 (OSF-2, Periostin) is a secreted extracellular matrix protein (ECM) expressed in fibroblasts during bone and teeth development. Expression of OSF-2 has been also related to the progression and drug resistance of different tumors. The present study investigated the role of OSF-2 by evaluating its expression in the primary serous ovarian cancer cell line, sensitive (W1) and resistant to doxorubicin (DOX) (W1DR) and methotrexate (MTX) (W1MR). The OSF-2 transcript (real-time PCR analysis), protein expression in cell lysates and cell culture medium (western blot), and expression of the OSF-2 protein in cell lines (immunofluorescence) were investigated in this study. Increased expression of OSF-2 mRNA was observed in drug-resistant cells and followed by increased protein expression in cell culture media of drug-resistant cell lines. A subpopulation of ALDH1A1-positive cells was noted for W1DR and W1MR cell lines; however, no direct co-expression with OSF-2 was demonstrated. Both drugs induced OSF-2 expression after a short period of exposure of the drug-sensitive cell line to DOX and MTX. The obtained results indicate that OSF-2 expression might be associated with the development of DOX and MTX resistance in the primary serous W1 ovarian cancer cell line.

scholarly journals Dissecting Mechanisms of Melanoma Resistance to BRAF and MEK Inhibitors Revealed Genetic and Non-Genetic Patient- and Drug-Specific Alterations and Remarkable Phenotypic Plasticity

Cells ◽  
2020 ◽  
Vol 9 (1) ◽  
pp. 142 ◽  
Author(s):  
Mariusz L. Hartman ◽  
Malgorzata Sztiller-Sikorska ◽  
Anna Gajos-Michniewicz ◽  
Malgorzata Czyz
Keyword(s):  
Phenotypic Plasticity ◽  
Cell Lines ◽  
Mapk Pathway ◽  
Acquired Resistance ◽  
Resistance Mechanisms ◽  
Genetic Alterations ◽  
Resistant Cell ◽  
Cross Resistance ◽  
Preclinical Model ◽  
Development Of Resistance

The clinical benefit of MAPK pathway inhibition in BRAF-mutant melanoma patients is limited by the development of acquired resistance. Using drug-naïve cell lines derived from tumor specimens, we established a preclinical model of melanoma resistance to vemurafenib or trametinib to provide insight into resistance mechanisms. Dissecting the mechanisms accompanying the development of resistance, we have shown that (i) most of genetic and non-genetic alterations are triggered in a cell line- and/or drug-specific manner; (ii) several changes previously assigned to the development of resistance are induced as the immediate response to the extent measurable at the bulk levels; (iii) reprogramming observed in cross-resistance experiments and growth factor-dependence restricted by the drug presence indicate that phenotypic plasticity of melanoma cells largely contributes to the sustained resistance. Whole-exome sequencing revealed novel genetic alterations, including a frameshift variant of RBMX found exclusively in phospho-AKThigh resistant cell lines. There was no similar pattern of phenotypic alterations among eleven resistant cell lines, including expression/activity of crucial regulators, such as MITF, AXL, SOX, and NGFR, which suggests that patient-to-patient variability is richer and more nuanced than previously described. This diversity should be considered during the development of new strategies to circumvent the acquired resistance to targeted therapies.

Novel Targeting Of Phospho-Marcks Overcomes Drug Resistance and Induces Anti-Tumor Activity In Preclinical Models Of Multiple Myeloma

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 282-282 ◽  
Author(s):  
Yijun Yang ◽  
Manujendra N Saha ◽  
Yan Chen ◽  
Lugui Qiu ◽  
Donna E Reece ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Drug Resistance ◽  
Cell Lines ◽  
Research Funding ◽  
Resistant Cell ◽  
Drug Resistant ◽  
Sensitive Cell ◽  
Bortezomib Treatment ◽  
Cell Cycle Pathway ◽  
Rpmi 8226

Abstract Multiple myeloma (MM) remains incurable due to the development of a drug-resistant phenotype after prolonged therapy. Myristoylated alanine-rich C-kinase substrate (MARCKS) is a protein kinase C (PKC) substrate that plays an important role in cell adhesion, spreading and invasion. Our previous studies found that overexpression of phospho-MARCKS (pMARCKS) was detected in developed drug resistant MM cell lines (RPMI-8226 R5, MM.1R) relative to their parental drug sensitive cell lines (RPMI-8226S, MM.1S). We hypothesized that pMARCKS is involved in chemo- and novel drug resistance in MM. To further evaluate the drug resistance, we exposed both RPMI-8226 R5 and MM.1R cell lines to varying dosages of bortezomib, dexamethasone, doxorubicin, and lenalidomide. By MTT assay, both resistant cell lines were found to have significantly higher viability to all 4 drugs compared to their respective non-resistant lines. In addition, Western blot analysis showed increased pMARCKS expressions in all 3 bortezomib resistant cell lines 8226.BR, OPM2.BR, and ANBL-6.BR as compared to their respective bortezomib sensitive cell lines. We next acquired MM patient samples collected at diagnosis and at relapse after bortezomib treatment, and investigated their pMARCKS expression with immunoblotting analyses. The patient samples collected from relapse after bortezomib treatment had higher pMARCKS expression than those collected at diagnosis. Furthermore, we studied additional 3 primary MM patient samples with high pMARCKS expressions and 3 with low expressions for their vaibility after a 36 hour bortezomib treatment, and found that the samples with high pMARCKS expressions were more resistant to bortezomib than those with low pMARCKS expressions (mean IC50 of 7.1 nM and mean IC50 of 4.8 nM, respectively; p = 0.042). Importantly, combination of a low dosage of bortezomib (5.0 nM) with either 2.5 uM or 5.0 uM of enzastaurin (an inhibitor of phospho-PKC), displayed a synergistic cytotoxicity on myeloma cells with high pMARCKS expressions. To further elucidate the role of pMARCKS in drug resistance, we knocked down pMARCKS expression by transfecting siMARCKS into 8226 R5 and MM.1R cells. Following the knockdown, both cell lines had significantly lower viability after treatment with either bortezomib, dexamethasone, doxorubicin, or lenolidomide, in comparison to empty vector controls. FACS analysis and annexin V assay of the knockdown cells and the control cells from both cell lines showed significantly induction of G1/S cell cycle arrest and apoptosis in the knockdown cells. The immunoprecipitation (IP) and chromatin immunoprecipitation (ChIP) DNA-qPCR analysis further demonstrated that pMARCKS regulates SKP2 expression through binding with E2F1, mediating SKP2/p27Kip1 cell cycle pathway. Finally, we investigated the effect of inhibition of pMARCKS in a 8226 R5 xenograft model of SCID mice. Mice injected with shMARCKS-transfected 8226 R5 cells and received bortezomib showed significant retardation of tumor growth and prolonged survival compared to the control groups. Taken together, our data indicate that pMARCKS is constitutively activated in resistant and relapsed MM cells and contributes to drug resistance by regulating E2F1 mediated SKP2/p27Kip1 cell cycle pathway, thus providing a preclinical rationale for targeting pMARCKS as a promising approach in patients with refractory/relapsed MM. Disclosures: Reece: BMS: Research Funding; Celgene: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Merck: Honoraria, Research Funding; Millennium: Research Funding; Novartis: Honoraria; Onyx: Honoraria.

Biopsy-driven study to identify biomarkers of drug resistance in patients with triple-negative breast cancer.

2012 ◽  
Vol 30 (30_suppl) ◽  
pp. 87-87
Author(s):  
Adriana Aguilar-Mahecha ◽  
Josiane Lafleur ◽  
Elaheh Ahmadzadeh ◽  
Ewa Przybytkowski ◽  
Carole Seguin ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Drug Resistance ◽  
Cell Lines ◽  
Triple Negative ◽  
Acquired Resistance ◽  
Great Difficulty ◽  
Clinical Samples ◽  
Drug Resistant ◽  
Breast Cancers ◽  
Breast Cancer Patients

87 Background: Resistance to chemotherapy is the underlying cause of death in most patients dying of breast cancer. Patients with early stages of breast cancer whose tumor is or becomes resistant to chemotherapy have a poor prognosis, while women with advanced breast cancer live as long as their tumors respond to chemotherapy. Because of the great difficulty of obtaining clinical samples from drug resistant tumors in patients, there is scant information about molecular factors from actual drug resistant tumors. This project aims to systematically profile resistant triple negative breast cancers (TNBCs) in order to discover molecular “resistance” genes/proteins as a first step to develop strategies to overcome drug resistance. Methods: Paired biopsies are collected from TNBC patients (NCT01276899). Four needle core biopsies are collected before the initiation of treatment and 2 weeks before surgery or at the time of progression in the neoadjuvant and metastatic settings respectively. Paired biopsies will undergo Next Gen Sequencing, flow sorted aCGH analysis, gene expression and miRNA profiling as well as phosphoproteomic profiling using reverse phase protein arrays. Results: We have currently enrolled 28 patients in the neoadjuvant setting and 3 metastatic patients. We have standardized the methods of collection and processing of tissue and blood specimens to ensure their molecular integrity and compatibility with different genomic and proteomic molecular platforms. Analysis of tumor cellularity has been incorporated into our quality control and we have optimized the extraction of nucleic acids to obtain high yields and optimal quality. In parallel, we have generated acquired resistance to paclitaxel in a panel of TNBC cell lines. These cell lines will also undergo genomic profiling and exome sequencing to identify molecular markers of resistance that will be correlated with the markers found in patient samples. Conclusions: This project will allow us to identify the molecular factors responsible for drug resistance in TNBCs and enable the elaboration of strategies to overcome resistance.

scholarly journals Identifying Mechanisms Associated with Venetoclax Resistance in Multiple Myeloma (MM)

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2668-2668
Author(s):  
Yuan Xiao Zhu ◽  
Laura Ann Bruins ◽  
Joseph Ahmann ◽  
Cecilia Bonolo De Campos ◽  
Esteban Braggio ◽  
...  
Keyword(s):  
Cell Line ◽  
Board Of Directors ◽  
Cell Lines ◽  
Acquired Resistance ◽  
Resistant Cell ◽  
Resistant Cell Line ◽  
Sensitive Cell ◽  
Advisory Committees ◽  
Bcl2 Family ◽  
Family Regulation

Abstract Venetoclax (VTX) is a selective small-molecule inhibitor of BCL-2 that exhibits antitumoral activity against MM cells presenting lymphoid features and those with translocation t(11;14). Despite its impressive clinical activity, VTX therapy for a prolonged duration can lead to drug resistance. Therefore, it is important to understand the underlying mechanisms of resistance in order to develop strategies to prevent or overcome resistance. In the present study, we established four VTX resistant human myeloma cell lines (HMCLs) from four sensitive HMCLs, including three with t(11;14), in culture with a stepwise increase in treatment dose with VTX. To identify the molecular basis of acquired VTX resistance, whole exon sequencing (WES), mRNA-sequencing (mRNAseq), and protein expression assays were performed in the four isogenic VTX-sensitive/resistant HMCLs and three MM patients with samples collected before VTX administration and after clinical resistance to the drug. Compared with sensitive cell lines and patient samples collected before VTX administration, mRNAseq analysis identified downregulation of BIM and upregulation of BCLXL in both resistant cell lines and MM cells from relapse patients. Other transcriptional changes detected included upregulation of AURKA, BIRC3, BIRC5, and IL32. Enrichment analysis of differentially expressed genes suggested involvement of PI3K and MAPK signaling, likely associated with cytokines, growth factors (EGF, FGF and IGF family members), and receptor tyrosine kinase (EGF and FGF). Western blot analysis was performed to compare BCL2 family expression in resistant cell lines versus sensitive cell lines and it showed upregulation of BCL2 survival members (such as MCL-1 and BCLXL), and downregulation of pro-apoptotic BH3 members (such as BIM and PUMA). BIM expression was completely lost in one resistant cell line, and introduction of exogenous BIM into this cell line enhanced VTX sensitivity. Interestingly, BCL2 was upregulated in some resistant cell lines generated after a long-term treatment with VTX, suggesting BCL2 expression level may not be suitable as a marker of VTX sensitivity for acquired resistance. Unlike in CLL, BCL2 mutations were not identified through WES in any resistant cell lines or primary patient sample harvested after relapse. While 8 genes were mutated in two resistant samples , no clear mutational pattern emerged . Based on the above, we further tested some specific inhibitors in in vitro or ex vivo cell models to help understanding resistant mechanism and identify strategies to overcome VTX resistance. We found that inhibition of MCL-1, with the compound S68345, substantially enhanced VTX sensitivity in three resistant HMCLs and in primary cells from one relapsed MM patient. A BCLXL inhibitor (A155463) only significantly enhanced VTX sensitivity in one resistant cell line after co-treatment with VTX. Co-treatment of the other three resistant cell lines with VTX, S68345 and A155463 resulted in the most synergistic anti-myeloma activity, suggesting those cell lines are co-dependent on MCL-1, BCLXL, and BCL2 for survival, although they are more dependent on MCL-1. We also found that inhibition of PI3K signaling, IGF1, RTK (EGF and FGF) and AURKA significantly increased VTX sensitivity, partially through downregulation of MCL-1, and BCLXL, and upregulation of BIM. Conventional anti-MM drugs such as dexamethasone, bortezomib and lenalidomide, were shown to have little activity on augmenting VTX sensitivity in most resistant cell lines. In summary, we find that acquired resistance to VTX in MM is largely associated with BCL2 family regulation, including upregulation of survival members such as MCL-1, BCLXL, BCL2, and downregulation of pro-apoptotic members, especially BIM. Our study also indicates that upstream signaling involved in BCL2 family regulation during acquired resistance is likely related to cytokine, growth factor, and/or RTK-induced cell signaling such as PI3K. Co-inhibition of MCL-1, or BCLXL, as well as the upstream PI3K, RTK (FGF and EGF), IGF-1 mediated signaling were effective in overcoming VTX resistance. Disclosures Fonseca: Mayo Clinic in Arizona: Current Employment; Amgen: Consultancy; BMS: Consultancy; Celgene: Consultancy; Takeda: Consultancy; Bayer: Consultancy; Janssen: Consultancy; Novartis: Consultancy; Pharmacyclics: Consultancy; Sanofi: Consultancy; Merck: Consultancy; Juno: Consultancy; Kite: Consultancy; Aduro: Consultancy; OncoTracker: Consultancy, Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy; AbbVie: Consultancy; Patent: Prognosticaton of myeloma via FISH: Patents & Royalties; Scientific Advisory Board: Adaptive Biotechnologies: Membership on an entity's Board of Directors or advisory committees; Caris Life Sciences: Membership on an entity's Board of Directors or advisory committees.

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