Stearoyl CoA Desaturase 1 (SCD1) Is Upregulated in Rapidly Growing Myeloma Cells and Is Required for Cell Proliferation,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3943-3943 ◽  
Author(s):  
Sathisha Upparahalli Venkateshaiah ◽  
Sharmin Khan ◽  
Wen Ling ◽  
Linda Saint John ◽  
Rakesh Bam ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Cell Proliferation ◽  
High Risk ◽  
Rapid Growth ◽  
Conflicts Of Interest ◽  
Saturated Fatty Acids ◽  
Molecular Signature ◽  
Myeloma Cells ◽  
Stearoyl Coa Desaturase

Abstract Abstract 3943 Myeloma plasma cell high labeling index and molecular signature of proliferation are strong adverse prognostic factors often characterize patients with high risk disease. The overall aim of the study was to identify cell proliferation associated genes implicating highly proliferating myeloma cells in the supportive bone marrow environment. To shed light on molecular factors associated with rapid growth of myeloma cells, primary myeloma cells from 10 patients, molecularly classified as high risk were engrafted in SCID-rab mice. Growth rate of myeloma varied between patients' cells but in all cases myeloma propagated within and surrounding the supportive implanted bone but not in any murine organs. We performed global gene expression profiling (GEP) on myeloma plasma cells recovered from mice and compared their GEP with the baseline, pre-injected myeloma cells. Based on stringent criteria (e.g. p<0.05, >2 folds) approximately 127 probe sets were commonly overexpressed and 36 probe sets underexpressed in myeloma cells from SCID-rab mice than baseline myeloma cells. Genes whose expression altered were mainly associated with proliferation, survival, metabolism, transcription and immunity. Among genes involved in cell proliferation we indentified stearoyl CoA desaturase 1 (SCD1), which was upregulated in 7 of 10 cases by overall 2.3±0.6 folds (p<0.01). In coculture of primary myeloma cells with the supportive osteoclasts (n=8), SCD1 was upregulated in 6 of 8 cocultures by 5.6±2.4 folds (p<0.02). SCD1 upregulation in vivo and in cocultures was consistently observed in 3 different GEP probe sets. SCD1 is a rate-limiting enzyme responsible for synthesis of monounsaturated fatty acids and is activated in highly proliferating tumor cells to sustain the increasing demand of new membrane phospholipids and energy storage, and reducing intracellular content of cytotoxic saturated fatty acids. Various SCD1 inhibitors are currently being evaluated for metabolic diseases. In vitro, small molecule SCD1 inhibitor (BioVision) dose dependently (0.1–10 μM, 96 hrs) inhibited growth of rapidly growing myeloma cell lines (n=5) but had moderate inhibitory effect on their survival. Compared to control vehicle-treated cultures, numbers of viable myeloma cells were reduced by 76±5% (p<0.008) and 51±3% (p<0.0001) following treatment with 0.1 μM and 5 μM of SCD1 inhibitor, respectively. Cell viability was reduced from 91±0.5% in control groups to 82±3% (p<0.05) and 73±5% (p<0.03) in cultures treated with 0.1 μM and 5 μM of SCD1 inhibitor, respectively. In vivo, luciferase-expressing H929 myeloma cells were engrafted in SCID-rab mice. Myeloma growth was monitored by live-animal bioluminescence imaging. Upon establishment of myeloma hosts were treated with SCD1 inhibitor using Alzet osmotic pump directly connected to the open side of the implanted bone and constantly released drug (1.25 μg/hour) or vehicle over a period of 2 weeks. At experiment's end myeloma burden was increased from pretreatment levels by 49±3 folds and 30±3 folds in control vehicle- and SCD1 inhibitor-treated hosts, respectively (p<0.01). We conclude that SCD1 is highly activated in proliferating myeloma cells and is essential for their rapid growth. Disclosures: No relevant conflicts of interest to declare.

FOXM1, CDK6 and Rb Dependent Drug Resistance and Senescence in Myeloma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4456-4456 ◽  
Author(s):  
Chunyan Gu ◽  
Ruini Chen ◽  
Xuefang Jing ◽  
Siegfried Janz ◽  
Ye Yang
Keyword(s):  
Drug Resistance ◽  
High Risk ◽  
Conflicts Of Interest ◽  
Expression Profiles ◽  
Physical Interaction ◽  
Mrna Levels ◽  
Agar Culture ◽  
Myeloma Cells

Abstract We recently reported that FOXM1 is a promising therapeutic target in multiple myeloma (MM), particularly for the subset of patients with high-risk disease.Because high-risk myeloma exhibits a strong predilection to early drug-resistant relapse following first-line therapy, we here decided to evaluate the role of FOXM1 in the acquisition of drug resistance by myeloma cells. We analyzed gene expression profiles of 88 paired myeloma samples at baseline and relapse from the UAMS Total Therapy 2 cohort and found that FOXM1 mRNA levels were significant upregulated in relapsed myeloma and that this was associated with poor event-free and overall survival. Laboratory studies showed that enforced expression of FOXM1 in human myeloma cell lines (HMCLs) results in decreased sensitivity of cells to widely used myeloma drugs, such as bortezomib and doxorubicin. This was observed in vitro, in both bulk cell and soft-agar culture, and in vivo using xenografting in mice. Biochemical analysis of HMCLs revealed physical interaction of FOXM1 with CDK6 and Rb, key regulators of cell cycle progression and cellular senescence, respectively.Treatment with small-compound CDK6 inhibitor, inhibited myeloma growth, decreased clonogenicity of myeloma, and ameliorated FOXM1-dependent senescence. Genetic and pharmacological targeting of FOXM1 in myeloma cells using shRNA and thiostreptone respectively, led to growth arrest and senescence, while elevated expression of FOXM1 reversed these phenotypes. In sum, our findings implicating the FOXM1-CDK6-Rb network in drug resistance and senescence of high-risk myeloma point to new treatment opportunities for this difficult-to-cure neoplasm. Disclosures No relevant conflicts of interest to declare.

scholarly journals Stearoyl-CoA desaturase-1 impairs the reparative properties of macrophages and microglia in the brain

2020 ◽  
Vol 217 (5) ◽  
Author(s):  
Jeroen F.J. Bogie ◽  
Elien Grajchen ◽  
Elien Wouters ◽  
Aida Garcia Corrales ◽  
Tess Dierckx ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Ex Vivo ◽  
Saturated Fatty Acids ◽  
Monounsaturated Fatty Acids ◽  
Demyelinating Diseases ◽  
Efflux Transporter ◽  
Stearoyl Coa Desaturase ◽  
Phenotypic Shift ◽  
The Brain

Failure of remyelination underlies the progressive nature of demyelinating diseases such as multiple sclerosis. Macrophages and microglia are crucially involved in the formation and repair of demyelinated lesions. Here we show that myelin uptake temporarily skewed these phagocytes toward a disease-resolving phenotype, while sustained intracellular accumulation of myelin induced a lesion-promoting phenotype. This phenotypic shift was controlled by stearoyl-CoA desaturase-1 (SCD1), an enzyme responsible for the desaturation of saturated fatty acids. Monounsaturated fatty acids generated by SCD1 reduced the surface abundance of the cholesterol efflux transporter ABCA1, which in turn promoted lipid accumulation and induced an inflammatory phagocyte phenotype. Pharmacological inhibition or phagocyte-specific deficiency of Scd1 accelerated remyelination ex vivo and in vivo. These findings identify SCD1 as a novel therapeutic target to promote remyelination.

COVID-19: Opinion in Prevention and dietary based management hypothesis

Coronaviruses ◽  
2020 ◽  
Vol 01 ◽  
Author(s):  
Ashraf Talaat Youssef
Keyword(s):  
Fatty Acids ◽  
Saturated Fatty Acids ◽  
Lung Damage ◽  
Gastric Aspirate ◽  
Enveloped Viruses ◽  
Multiple Organs ◽  
Fold Reduction ◽  
Antiviral Activities

The pandemic of COVID-19 had started in Wuhan city china in late 2019 with a subsequent worldwide spread. The viral infection can seriousely affect multiple organs mainly lungs, kidneys, heart, liver and brain and may lead to respiratory, renal, cardiac or hepatic failure.Vascular thrombosis of unexplained mechanism that may lead to widespread blood clots in multiple organs and cytokine storms that result of overstimulation of the immune system subsequent of lung damage may lead to sudden decompensation due to hypotension and more damage to liver, kidney, brain or lungs.Until now no drug had proved efficient in getting rid of the problem and controlling the pandemic mainly depends on preventive measures.Many preventive measures can be considered to prevent the worldwide spread of viral transmission. Polyunsaturated long chain fatty acids (PUFAs) and the medium chain saturated fatty acids (MCSFAs) and their corresponding monoglycerides had high antiviral activities against the enveloped viruses which reach to more than 10,000 -fold reduction in the viral titres in vitro and in vivo after testing of its gastric aspirate, and can contribute to the systemic immunity against the enveloped viruses.

scholarly journals Polyunsaturated ω-3 fatty acids inhibit ACE2-controlled SARS-CoV-2 binding and cellular entry

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Anna Goc ◽  
Aleksandra Niedzwiecki ◽  
Matthias Rath
Keyword(s):  
Fatty Acids ◽  
Polyunsaturated Fatty Acids ◽  
Eicosapentaenoic Acid ◽  
Linolenic Acid ◽  
Cathepsin L ◽  
Saturated Fatty Acids ◽  
Monounsaturated Fatty Acids ◽  
In Vivo Experiments ◽  
Lipid Compounds

AbstractThe strain SARS-CoV-2, newly emerged in late 2019, has been identified as the cause of COVID-19 and the pandemic declared by WHO in early 2020. Although lipids have been shown to possess antiviral efficacy, little is currently known about lipid compounds with anti-SARS-CoV-2 binding and entry properties. To address this issue, we screened, overall, 17 polyunsaturated fatty acids, monounsaturated fatty acids and saturated fatty acids, as wells as lipid-soluble vitamins. In performing target-based ligand screening utilizing the RBD-SARS-CoV-2 sequence, we observed that polyunsaturated fatty acids most effectively interfere with binding to hACE2, the receptor for SARS-CoV-2. Using a spike protein pseudo-virus, we also found that linolenic acid and eicosapentaenoic acid significantly block the entry of SARS-CoV-2. In addition, eicosapentaenoic acid showed higher efficacy than linolenic acid in reducing activity of TMPRSS2 and cathepsin L proteases, but neither of the fatty acids affected their expression at the protein level. Also, neither reduction of hACE2 activity nor binding to the hACE2 receptor upon treatment with these two fatty acids was observed. Although further in vivo experiments are warranted to validate the current findings, our study provides a new insight into the role of lipids as antiviral compounds against the SARS-CoV-2 strain.

scholarly journals The Molecular Basis of Long First Remissions in Normal Karyotype AML Patients

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3827-3827
Author(s):  
Francesca Ferraro ◽  
Christopher A Miller ◽  
Amy Abdalla ◽  
Nichole Helton ◽  
Nathan Salomonis ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Somatic Mutations ◽  
Conflicts Of Interest ◽  
Normal Karyotype ◽  
High Dose ◽  
Intermediate Risk ◽  
The Mean ◽  
Cebpa Mutations

Currently, it is not clear why some patients with acute myeloid leukemia (AML) can be "cured" with chemotherapy alone; are they living with small amounts of disease that is held in check by immunologic (or other) mechanisms, or is their disease really eradicated? The percentage of cytogenetically normal AML patients who have long (>5 years) first remissions (LFRs) after chemotherapy alone is low (about 9.1% in patients <60 years and 1.6% in >60 years1). For this reason, most intermediate risk patients are offered allogeneic transplantation to decrease their risk for relapse. To better understand mechanisms of chemotherapy sensitivity in AML, we performed an analysis of the mutation landscape and persistence, using samples from 8 normal karyotype LFR patients (without CEBPA mutations) who received standard "7+3" induction and high dose cytarabine consolidation as their only therapy. The mean age at diagnosis was 43.5 years, and the mean follow up in first remission is 7.6 years; none of these patients has relapsed to date. For each case, we performed enhanced exome sequencing at diagnosis (235x coverage of the entire exome, and ~1008x coverage of recurrently mutated AML genes). Each case had at least one documented AML driver mutation, with a median of 29 somatic mutations in the exome space. We created probes for 225 mutations (mean 28 per case), and performed error-corrected sequencing (Haloplex) for all available remission samples. The mean depth of Haloplex coverage was 1607x, and each sample had at least one AML-specific mutation assayed, with a sensitivity of 1 cell in 1,750 (0.06%). 7/8 patients demonstrated complete clearance of all mutations in all remission samples tested, which was confirmed with digital droplet PCR for 5 cases, with a sensitivity of detection of 1 cell in 100,000. In one case, we detected a persistent ancestral clone harboring DNMT3AR882H, which can be associated with long first remissions for some patients2. Strikingly, the founding clone in all 8 cases had one or more somatic mutations in genes known to drive cell proliferation (e.g. MYC, FLT3, NRAS, PTPN11, Figure 1 top panel). These are usually subclonal mutations that occur late during leukemic progression, suggesting that the presence of a "proliferative hit" in the founding clone might be important for chemotherapy clearance of all the AML cells in a given patient. To support this hypothesis, we analyzed the mutational clearance of 82 AML cases with paired diagnosis and day 30 post-chemotherapy bone marrow samples. We observed that, whether present in the founding clone or in subclones, mutations in MYC, CEBPA, FLT3, NRAS, and PTPN11 cleared after induction chemotherapy in all samples, while other mutations were often persistent at day 30 (e.g. DNMT3A, IDH1, IDH2, NPM1, TET2; Figure 1 bottom panel). Compared to other published sequencing studies of AML, MYC and NRAS mutations were significantly enriched in this small cohort (MYC p= 0.002, and NRAS p= 0.034), with MYC enrichment being particularly striking (37.5% versus 1.8%). All MYC mutations were canonical single base substitutions occurring in the highly conserved MYC Box 2 domain at the N-terminus of MYC (p.P74Q or p.T73N). Overexpression of MYCP74Q in murine hematopoietic progenitors prolonged MYC half life (89 min vs. 44 min for wild type), and enhanced cytarabine sensitivity at all concentrations tested (range 10-1000 nM, p=0.0003), both in vitro and in a MYC-driven leukemia model in vivo. MYC expression measured with flow cytometry in the blasts of the LFR samples was significantly higher (p=0.045) compared to unfavorable risk (complex karyotype) or other intermediate risk categories, but similar to good risk AML (biallelic CEBPA mutations, core binding factor fusion-associated AML, and AML with isolated NPMc), suggesting that activation of the MYC pathway may represent a shared feature of chemosensitive patients. Taken together, these data suggest that some intermediate patients who are effectively "cured" with chemotherapy alone may not have persistent subclinical disease, nor retained ancestral clones that could potentially contribute to relapse. Importantly, these patients often have mutations driving cell proliferation in the founding clone, indicating that the presence of specific mutations in all malignant cells may be critical for complete AML cell clearance with chemotherapy. 1. Blood Adv. 2018 Jul 10; 2(13): 1645-1650 2. N Engl J Med 2018; 378:1189-1199 Disclosures No relevant conflicts of interest to declare.

RASGRP1/APTX Ratio Strongly Correlates with Clinical Response and Survival in AML Patients Treated with Tipifarnib-Bortezomib Combination.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1028-1028
Author(s):  
Stefania Paolini ◽  
Emanuela Ottaviani ◽  
Sarah Parisi ◽  
Federica Salmi ◽  
Barbara Lama ◽  
...  
Keyword(s):  
Overall Survival ◽  
Adverse Event ◽  
High Risk ◽  
Clinical Response ◽  
Stable Disease ◽  
Response Rate ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Secondary Aml

Abstract Abstract 1028 Poster Board I-50 Background: Outcome of elderly acute myeloid leukemia (AML) patients is dismal. Targeted-therapies might improve current results by overcoming drug-resistance and reducing toxicity. In particular, the farnesyl-transferase inhibitor Tipifarnib (Zarnestra®), and the proteasome inhibitor Bortezomib (Velcade®), appeared synergistic in AML cells ex vivo, and their association was shown to be safe in vivo in a phase I trial by our group. Aim We conduced a phase II study aiming to assess efficacy and toxicity of Tipifarnib-Bortezomib association in AML patients >18 years, unfit for conventional therapy, or >60 years, in relapse. Furthermore, we aimed to identify biological features potentially predictive of clinical response. In particular, we focused on the RASGRP1/APTX ratio, which was previously found to be effective in predicting treatment response in patients treated with Tipifarnib alone. Methods: Bortezomib (1.0 mg/m2) was administered as weekly infusion for three consecutive weeks (days 1, 8, 15). Tipifarnib was administered at dose of 300-600 mg BID for 21 consecutive days. Response was assessed at the end of each cycle (28 days). Patients' withdrawn was planned in case of progression or stable disease after six cycles. Real-time quantitative-PCR (q-PCR) was used for RASGRP1/APTX quantification. Results: Eighty patients were enrolled (47 male). Median age was 71 years (43-89) and WBC at diagnosis was 4.2 × 109/L (0.5- 42.1). Thirty-two out of 80 patients had a secondary-AML, 14 had a high risk cytogenetic and 42 were previously untreated. Seventy-five patients actually initiated the treatment, 62 completed at least the first cycle while 13 early dropped out for non-leukemia related adverse event. Nine patients achieved complete remission (CR), 1 patients obtained a partial response (PR) and in 2 cases an hematological improvement (HI) was documented for an overall response rate (ORR) of 19%. Eighteen had progressive disease (PD) and the remaining showed stable disease (SD). Median time to response was 112 days, corresponding to 4 cycles (range 2-14). Marrow response (CR+PR) was significantly associated with overall survival (OS) (p<0.0001). RASGRP1/APTX was evaluated before treatment initiation on bone marrow (BM) and/or peripheral blood (PB). The median RASGRP/APTX value on BM was 15.3 (15-19.8) in responder patients and 2.2 (0.5-25.9) in non responders, respectively (p=0.00006). Its median value on PB was 31.6 (19.3-35.5) in responders and 6.4 (0.5-27.1) in non responders, respectively (p=0.00001). Interestingly, no marrow responses were recorded in patients with marrow RASGRP1/APTX ratio <8, while the response rate was 43% in patients with RASGRP1/APTX >8 (p<0.0001). Finally, RASGRP1/APTX levels significantly correlated with OS (p=0.001) with a median OS of 490 days and 162 days in patients with RASGRP1/APTX >8 and <8 respectively. Conversely, there was no correlation between cytogenetics, secondary AML, previous treatment and response or overall survival. Toxicity was overall mild, the most common adverse event being febrile neutropenia. Permanent treatment interruption due to Tipifarnib-Bortezomib related adverse events occurred in 13/75 (17%) of patients. With a median follow-up of 122 days (range 9-737), 57/75 (76%) patients are dead and 18/75 (24%) are alive, six of which in CR. Conclusion: We conclude that the clinical efficacy of the combination Tipifarnib-Bortezomib was similar to what reported for Tipifarnib alone. However, noteworthy, we could confirm that the RASGPR1/APTX BM or PB level is an effective predictor of response. Though higher RASGRP1/APTX is relatively rare (∼10% of cases), Tipifarnib (±Bortezomib) may represent an important option in a subset of high risk/frail AML patients. Acknowledgments: Supported by BolognAIL, AIRC, European LeukemiaNET, COFIN, FIRB 2006, Fondazione del Monte di Bologna e Ravenna. Disclosures: No relevant conflicts of interest to declare.

CCR4:CCL17 Interaction Influences TLR-9 Mediated Cell Survival and Proliferation In Chronic Lymphocytic Leukemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3593-3593
Author(s):  
Sonal C. Temburni ◽  
Ryon M. Andersen ◽  
Luke Janson ◽  
Xiao-Jie Yan ◽  
Barbara Sherry ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Conflicts Of Interest ◽  
Dose Range ◽  
Serum Levels ◽  
Lymphocytic Leukemia ◽  
Novel Therapy

Abstract Abstract 3593 Unlike other hematologic disorders, chronic lymphocytic leukemia(CLL) exhibits remarkable heterogeneity in the rates of disease progression among cases. CLL cells survive by receiving signals from the microenvironment via various receptors: B-cell antigen receptor (BCR), Toll-like receptors (TLRs) and cytokine and chemokine receptors. We previously reported that CLL clones with somatically mutated IGHVs and high (≥30%) percentage of CD38 expressing cells have the highest percentage of CCR4-expressing cells. To further explore the functional contribution of the CCR4:CCL17 axis in CLL, we studied CCL17-induced chemotactic behavior in 16 CLL cases. In transwell cultures we observed a bimodal migratory response to CCL17 at 2 doses in a dose range of 0.78– 25ng/ml, in ~60% of cases; the remaining cases showed maximal migration at a single dose (1.56 or 3.12ng/ml). A comparison of phenotypes of the migrated and non-migrated cell populations was undertaken in 10 cases, analyzing CXCR3, CXCR4, CCR4 and CCR7 that are involved in homing of cells to sites favoring growth, and CD31, CD38 and CD69, activation related molecules. The migrated cells consistently showed significantly higher percentages and densities of CD38 expression than the non-migrated cells suggesting a role for CD38 in the CCR4-mediated downstream pathway. CCR4 ligand, CCL17, is constitutively expressed in the thymus and is produced by dendritic cells, endothelial cells, keratinocytes and fibroblasts, whereas CCL22 is produced by tumor cells and the tumor microenvironment. Serum levels of both these ligands in untreated patients were quantified by ELISA. CCL17 levels ranged between 45-1, 229 pg/ml in U-CLL cases (n=23) and between 43-1, 418 pg/ml in M-CLL cases (n=30). CCL22 levels ranged between 121-5, 497 pg/ml in U-CLL cases (n=23) and 409-5, 502 pg/ml in M-CLL cases (n=30). The percentages of CCR4- expressing B cells directly correlated with percentages of T cells expressing CCR4 in individual cases, whereas they inversely correlated with both, serum levels of CCL17 (p< 0.01) and CCL22 (p< 0.05). CCL17 produced by DCs in peripheral organs may exert an accessory role in BCR- and TLR-9-mediated immune responses in B cells. We therefore tested if CCL17 supported BCR- and TLR-mediated proliferative responses in a cohort of 31 (16 U-CLL and 15M-CLL) CLL cases. CCL17 augmented BCR-mediated B-cell proliferation in 9/16 (56%) U-CLL cases, but only in 3/15 (20%) M-CLL cases. On the other hand, CCL17 showed an additive effect in promoting TLR-9-mediated cell proliferation in 13/15 (87%) M-CLL cases at a dose of 2ng/nl (approximating that detected in serum); it also augmented TLR-9 mediated B cell proliferation in 6/16 U-CLL cases but at a 5-fold or higher dose (10-25 ng/ml). In a subset of this cohort (8 cases) CCL17-induced modulation of molecules involved in the apoptotic process was studied. We found upregulation of anti-apoptotic proteins Mcl-1 and Bcl2 and down-regulation of pro-apoptotic molecules Bim, PUMA, and Bid in 5 of these cases. The pro-survival effects of CCL17 were partially abrogated by the blocking anti-CCR4 mAb (1G1). Taken together, these findings suggest that CCL17 plays a role in modulating TLR-9-mediated signaling and migration in CLL. Therefore, inhibition of CCR4:CCL17 interaction in vivo represents a novel therapy by preventing migration of CLL cells towards an environment that promotes their survival. Disclosures: No relevant conflicts of interest to declare.

Carfilzomib and Bortezomib Containing Regimens Yield High Rates Of MRD Negative Autologous Hematopoietic Progenitor Cell (HPC) Grafts In Multiple Myeloma (MM) and High Risk Smoldering Myeloma (SMM)

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2030-2030
Author(s):  
Nishant Tageja ◽  
Neha Korde ◽  
Constance Yuan ◽  
Kristen Cole ◽  
Jennifer Hsu ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Stem Cell ◽  
High Risk ◽  
Tumor Cell ◽  
Peripheral Blood ◽  
Conflicts Of Interest ◽  
Hematopoietic Progenitor Cell ◽  
Myeloma Cells ◽  
Cd34 Cells ◽  
The Mean

Abstract Background Regimens incorporating modern anti-myeloma drugs, such as carfilzomib (CFZ) and bortezomib (BOR), produce rapid, deep and durable responses in newly diagnosed myeloma patients but their effect on collection of autologous HPC is not well known, including minimal residual disease (MRD) testing of stem cell grafts. Employing older induction regimens (such as VAD), less sensitive flow cytometry techniques detected circulating myeloma cells in 38-46% of autologous HPC grafts (Stewart, et al. JCO. 2001 and Bourhis, et al. Haematologica. 2007). We hypothesized that the use of modern CRd combination therapy including Carfilzomib (CFZ)-Lenalidomide (LEN)-Dexamethasone (DEX) would significantly lower the rates of HPC product contamination. Methods Thirty-six patients, including 29 with MM and 7 with high-risk SMM, underwent HPC mobilization and collection following induction with CRd (n=30), LEN-BOR-DEX (RVd, n=4), Cyclophosphamide-BOR-DEX (CyBorD, n=1) and Cyclophosphamide-BOR-Prednisone (CyBorP, n=1). For HPC mobilization, all patients received 5 days of filgrastim at 10-16 mcg/kg/dose. A combination of the patient’s weight and a peripheral blood CD34 count after 4 doses was used to determine the likelihood of collecting > 4 x106 CD34+ cells/ kg in a single apheresis procedure after a fifth filgrastim dose, according to a previously published algorithm from our institution. Only subjects predicted to require > 1 apheresis by the algorithm received Plerixafor (PLX) at 240 mcg/kg/dose on the fifth day along with the fifth filgrastim dose. HPC collection occurred on day 6, 8 hours after the last mobilizing agent(s) administration. Product contamination with myeloma cells (i.e. MRD status) was evaluated using multi-parameter flow cytometry with a minimum of 3 x 106 events obtained (sensitivity detection rate 1 x 10-5) to examine expression of 9 antigens by the plasma cells. Results The median age at mobilization was 56.2 years (range 40-73) and 19 (53%) were male. At the time of HPC collection, 20 (55%) patients were in sCR/CR/nCR, 11 (30%) had VGPR with 4 PR (11%) and 1 SD (3%). The mean CD34+ cells in the peripheral blood were 33/uL on day 5 and 55/uL on day 6 for the whole cohort. Thirteen (36%) patients did not need PLX. Interestingly, the mean CD34+ count dropped by a mean of 2% from D5 to D6 in patients not receiving PLX while, as expected, it increased by 304% in those who did. The median number of CD34+ cells collected was 6.86 million/kg (range 2.6-12.5) for the whole cohort, (6.6 million/kg without PLX and 7.52 million with PLX p=0.46). Thirty-three of 36 patients (92%) achieved a collection of > 4 million cells /kg in a single apheresis procedure. The 30 patients treated with CRd had a median of 5 (range = 3-7) prior cycles containing LEN with a median of 12 days (range 1-34) between mobilization and last LEN dose. Only 2 of 36 (5%) products were found to have evidence of tumor cell contamination (i.e. MRD positive) using sensitive multiparameter flow cytometry, one patient in PR after 6 cycles of CRd and a second patient in CR after 5 cycles of RVd. Conclusions Modern anti-myeloma therapies, such as CRd and RVd, allow adequate HPC collection in a single apheresis procedure in most cases and improve the quality of the HPC product with greatly reduced tumor cell contamination compared to historical controls. Indeed, 34/36 (94%) patients treated with modern anti-myeloma therapy collected an MRD negative HPC product. Future prospective studies are needed to assess whether autologous stem cell transplants (ASCT) using tumor-free HPC products collected in the era of modern induction therapies have better outcomes. Disclosures: No relevant conflicts of interest to declare.

EBV-Encoded Mirnas Facilitate Inflammation and Tumor Formation Through Both Intra- and Inter-Cellular Regulation In Vivo Mouse Model

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3735-3735
Author(s):  
Natsuko Yamakawa ◽  
Jun Ogata ◽  
Takashi Yahata ◽  
Jun Lu ◽  
Kazuaki Yokoyama ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Tumor Cells ◽  
Small Rnas ◽  
Conflicts Of Interest ◽  
Functional Characterization ◽  
Tumor Formation ◽  
Sequencing Analysis ◽  
Immunodeficient Mice

Abstract Introduction EB virus (EBV) is associated with heterogeneous lymphomas. Hodgkin's lymphoma (HL) cells are embedded in non-neoplastic bystanders: B, T cells, and macrophages. Without these bystander cells, the lymphoma cells are incapable of being engrafted in immunodeficient mice. In this context, the bystanders are tumor-supportive “inflammatory niche”. Recently, EBV-infected cells produce exosomes that contain EBV specifically encoded miRNAs (EBV-miRNAs). The miRNAs are transferred to cells, and involved in tumor metastasis. However, the detailed mechanism is unknown. Accordingly, we hypothesized that exosomal EBV-miRNAs might redirect tumor surrounding immune cells from tumor reactive into tumor-supportive “inflammatory niche”. Methods We evaluated the expression of EBV-miRNAs in EBV+HL clinical specimens by in situ hybridization, their functional characterization in vitro, and their effects on persistent infection and tumor development in vivo humanized NOG mice model. Moreover, in order to clarify its sorting mechanism, trans factor and cis factor which determined secreted and non-secreted miRNAs was analyzed by use of mass-spectrograhy and next-generation sequencing. Results and Discussion The EBV-miRNAs effects were potent on monocyte/macrophage Mo/Mf in inducing CD69, IL-10, and TNF, suggesting that EBV-miRNAs might polarize Mo/Mf into tumor associated Mf (TAM). EBV-miRNAs suppress tumor cell proliferation in vitro, implying that it works as tumor-suppressor in the tumor cells, while they are required to develop LPD in vivo, which seems contradict to the result in vitro. These results suggest that EBV-miRNAs intra-cellularly regulate the tumor cells to adjust to the surrounding circumstances, for example, to escape from immune surveillance, and inter-cellularly regulate Mo/Mf to support the tumor survival or development. Most importantly, exosomal EBV-miRNAs derived from the tumor cells were transferred to Mf in human EBV+ HL samples. Interestingly, one EBV coded miRNA was not secreted at all, though it abundantly expresses in the cells. The miRNA has been reported to strongly promote cell proliferation in EBV infected tumor cells. It made us hypothesized that the sorting system of secretary and non-secretary miRNAs is critical in the formation of “inflammatory niche”. In order to clarify the mechanism of the sorting, the chimeric miRNA was constructed then, we determined the sequence, which regulates secretion and non-secretion, and purified the protein complex, which specifically bound to the sequence. Mass spectrography and successive knockdown assay, the trans factor which inhibits secretion was identified. Moreover, the next sequencing analysis for the small RNAs revealed that abundant EBV-coded small RNAs occupied RNA-induced silencing complex (RISC), and that non-secreted EBV-miRNA was specifically modified. It is now under investigation whether the modification is involved in the sort mechanism between secretary and non-secretary miRNAs. Taken together, EBV-miRNAs have critical roles in intra- and inter-cellular manner. Especially, the functions as an inter-cellular communicator might be important in the tumor formation and the mechanism needs further investigation. Disclosures: No relevant conflicts of interest to declare.

Combined Inhibition of Wee1 and Poly(ADP Ribose) Polymerase1/2 Synergistically Inhibits Acute Myeloid Leukemia Cells By Enhancing DNA Damage and the Induction of Apoptosis

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3621-3621 ◽  
Author(s):  
Jonathan C Snedeker ◽  
Tamara M Burleson ◽  
Raoul Tibes ◽  
Christopher C. Porter
Keyword(s):  
Flow Cytometry ◽  
Cell Proliferation ◽  
Dna Damage ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Combination Index ◽  
Annexin V ◽  
Concomitant Treatment ◽  
Western Blots

Abstract Introduction: Successful treatment of AML remains dependent upon cytotoxic chemotherapy. However, traditional regimens are not well tolerated by older patients who are at highest risk of disease, and salvage rates after relapse are low, necessitating novel therapeutic strategies. Our groups identified Wee1 as a potential therapeutic target in AML, particularly in the context of concomitant treatment with cytarabine (Tibes et al, Blood, 2012; Porter et al, Leukemia, 2012). Wee1 inhibits CDK1&2 via phosphorylation thereby stalling cell cycle progression. One consequence of Wee1 inhibition/CDK1 activation is impairment of DNA repair via homologous recombination (Krajewska et al, Oncogene, 2013). Cells in which HR is impaired are dependent upon Parp1/2 function, and HR deficient cells are particularly sensitive to Parp1/2 inhibition. Therefore, we hypothesized that combined Wee1 and Parp1/2 inhibition may result in greater inhibition of AML cell proliferation and survival than either alone. Methods: Human AML cell lines, MV4-11 and Molm-13, and a mouse AML that expresses MLL-ENL/FLT3-ITD were cultured with various concentrations of a Wee1 inhibitor (AZ1775) and a Parp1/2 inhibitor (olaparib) and counted 72 hours later by propidium iodide exclusion and flow cytometry. In some experiments, cells were split into fresh media to recover for 72 more hours. Combination Index (CI) values were calculated by the method of Chou and Talalay. Apoptosis was measured using Annexin V/7AAD and flow cytometry. Western blots were used to confirm inhibition of CDK1/2 phosphorylation and to measure DNA damage induction (gamma-H2AX). Results: Combined inhibition of Wee1 and Parp1/2 was synergistic, as measured by cell numbers at 72 hours, in all 3 cell lines tested, with combination index values ranging from 0.3 to 0.9. When cells were allowed to recover after treatment, those treated by single agents were able to continue proliferating. However, those treated with the combination did not recover as well or at all, indicating greatly impaired proliferative capacity. Combined inhibition of Wee1 and Parp1/2 also resulted in a significant increase in apoptosis greater than either drug alone. Western blots for gamma-H2AX confirmed that the combination of Wee1 and Parp1/2 resulted in more DNA damage than either drug alone. Discussion: Combined inhibition of Wee1 and Parp1/2 results in greater inhibition of AML cell proliferation, DNA damage and apoptosis than either drug alone. Future studies will include experiments with primary patient samples, as well as in vivo trials combining Wee1 inhibition with Parp1/2 inhibition. These preliminary studies raise the possibility of rational combinations of targeted agents for leukemia in those for whom conventional chemotherapeutics may not be well tolerated. Disclosures No relevant conflicts of interest to declare.

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