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.

Irinotecan Resistance – In Search of New Markers in CRC.

2021 ◽  
Author(s):  
Jakub Kryczka ◽  
Joanna Boncela
Keyword(s):  
Drug Resistance ◽  
Cell Lines ◽  
Expression Profiles ◽  
Resistance Mechanism ◽  
Interaction Network ◽  
In Vitro Study ◽  
Cancer Drug ◽  
New Genes

Abstract Colorectal cancer (CRC) is one of the most prominent causes of cancer death worldwide. Chemotherapeutic regimens consisting of different drugs combinations such as 5-fluorouracil, and oxaliplatin (FOLFOX) or irinotecan (FOLFIRI) have been proven successful in the treatment of CRC. However, chemotherapy often leads to the acquisition of cancer drug resistance followed by metastasis and in the aftermath therapeutic failure. The molecular mechanism responsible for drug resistance is still unclear. The systemic search for new biomarkers of this phenomenon may identify new genes and pathways. To understand the drug resistance mechanism in CRC, the in vitro study based on the molecular analysis of drug-sensitive cells lines vs drug-resistant cells lines has been used. In our study to bridge the gap between in vitro and in vivo study, we compared the expression profiles of cell lines and patient samples from the publicly available database to select the new candidate genes for irinotecan resistance. Using The Gene Expression Omnibus (GEO) database of CRC cell lines (HT29, HTC116, LoVo, and their respective irinotecan-resistant variants) and patient samples (GSE42387, GSE62080, and GSE18105) we compared the changes in the mRNA expression profile of the main genes involved in irinotecan body’s processing, such as transport out of the cells and metabolism. Furthermore, using a protein-protein interaction network of differently expressed genes between FOLFIRI resistant and sensitive CRC patients, we have selected top networking proteins (upregulated: NDUFA2, SDHD, LSM5, DCAF4, and COX10, downregulated: RBM8A, TIMP1, QKI, TGOLN2, and PTGS2). Our analysis provided several potential irinotecan resistance markers, previously not described as such.

Chronic Lymphocytic Leukemia-Associated Macrophages: Not Just Nurse Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 46-46
Author(s):  
Loic Ysebaert ◽  
Mary Poupot ◽  
Yovan Sanchez-Ruiz ◽  
Camille Laurent ◽  
Guy Laurent ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Conditioned Medium ◽  
Apoptotic Cell ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Lymphocytic Leukemia ◽  
Functional Interpretation

Abstract Abstract 46 Introduction: CLL cells interact with many accessory cells in an environment mimicking that of normal mature B cells. Role of antigen, cytokines, adhesion pathways are critical for many aspects in the disease course (proliferation/survival, migration or homing, drug resistance, and presumably relapse). Nurse-like cells (NLC) belong to a monocytic-derived, bystander population among CLL lymph node and spleen stromal cells. Aim: To investigate the nature, functions, and location of NLC within CLL microenvironment. Methods: Gene expression profiles (GEP) from in vitro expanded NLC from patients (n=10) were produced and compared to those from normal CD14+ monocytes, M1-polarized macrophages, M2-polarized macrophages and tumor-associated macrophages (produced in the lab or downloaded from GEO datasets). Principal Component Analysis was used to categorize these five populations of cells and in-house-built GSEA software was used for functional interpretation of their relevant gene lists. Protein expression patterns were validated with multi-analyte ELISArray kits, proteome profiler arrays, flow cytometry (FC) or immunohistochemistry (IHC). Results: New insights into the physiopathological role of NLC in CLL are suggested from five lines of evidence: 1/a Òmonocytic gene signatureÓ (i.e. a set of 549 genes) is shared by the NLC and the monocyte subtypes. The genes over-represented in NLC vs normal monocytes pinpointed positive modulation of apoptotic cell clearance (scavenger, mannose and complement receptors, LXRalpha), lipid metabolism (Apolipoprotein E, PPAR signaling), extracellular matrix-receptor interactions (integrins, SPARC, Matrix MetalloProteinases) and actin cytoskeleton remodeling. 2/unsupervised clustering show that NLC represent an M2-skewed, TAM-like cell population. They down-regulate mRNA and proteins for classic M1 inflammatory markers (e.g. IL-1, IL-6, IL-12, COX2) while increase secretion of TGFbeta, IL-10, CCL17 and CCL22 soluble factors. 3/these and previously published observations suggest that B-CLL-to-NLC interactions may orchestrate immunosuppression in this disease. PBMCs from Òwatch and waitÓ CLL patients (all stage A/Rai 0, mutated IgVH, low risk cytogenetics profile) or healthy donors were stimulated with anti-CD3/CD28 beads + IL-2, either in standard RPMI+10% FCS or in conditioned medium (CM, after 14d CLL-NLC co-culture in vitro) and their proliferation/phenotype were compared after 2 weeks. Significant expansion of T cells with Treg (CD4+CD25+FoxP3+) phenotype was observed only from CLL PBMCs grown in conditioned medium (mean % Treg: 2.85 vs 3.05 in CM for normal PBMCs, and 1.54 vs 15.9 in CM for CLL PBMCs, P< 0.05). 4/although NLC make immune synapses with live B-CLL, they do not phagocytose them. Over-expression of CD47 (ÒdonÕt eat meÓ signal) by B-CLL cells (mfi= 3490 vs 2581 on normal cells, P< 0.05, n=18) may provide them with a protective signal against NLC. 5/from our GEP, flow cytometric and IHC analyses, we propose CD163 (classic M2 marker) as a reliable tool to identify NLC in vivo. Although in vitro, CLL cells can pervert healthy donor monocytes into NLC, only CLL-derived NLC are truly CD14+ CD163+. In vivo, CD163 staining reveals putative NLC in CLL lymph nodes(LN)/spleen sections but not in bone marrow. In LN from all patients, NLC reside in the subcapsular areas and line vessel structures, suggesting a role in CLL cells trafficking. Most interestingly, NLC infiltrate pseudofollicles structures only in a subset of cases. We will present updated IHC and clinical presentation correlation studies. Conclusions: Our results suggest that the role of NLC in CLL might be broader than initially thought. Beside of nursing and conferring drug resistance, NLC may also be crucial in the setting of immunosuppression, of CLL cells recruitment, and should thus be considered as therapeutic targets. Disclosures: Off Label Use: GA101 is not currently approved for CLL treatment.

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.

A Whole Genome In Vivo Crispr Screen in Primary ALL Predicts Leukaemic Relapse

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2619-2619
Author(s):  
Katherine Dormon ◽  
Elda S Latif ◽  
Matthew Bashton ◽  
Deepali Pal ◽  
Matthew Selby ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Conflicts Of Interest ◽  
Gene Set Enrichment Analysis ◽  
Whole Genome ◽  
Competitive Situation ◽  
Nsg Mice ◽  
A Genome ◽  
Crispr Screen

Abstract Although paediatric acute lymphoblastic leukaemia (ALL) has a favourable prognosis, a number of cases will invariably relapse. One of the major problems associated with relapse is drug resistance, in particular to glucocorticoids, the mainstay of ALL treatment. Examining the underlying mechanisms is complicated by clonal heterogeneity within a patient and the potential impact of the leukaemic niche. To address mechanisms of drug resistance in a patient-relevant setting, we performed a genome-wide in vivo CRISPR screen in primary ALL material. To that end, we took advantage of primografted material from patient L707, who initially presented with a Dexamethasone (DEX) sensitive t(17;19) ALL, but relapsed 5 months after initial diagnosis. We transduced DEX sensitive presentation cells with the full genome GeCKOv2 CRISPR library, before transplantation into immunodeficient NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. Mice were subsequently treated with DEX by oral gavage (15mg/kg for 5 weeks, 10mg/kg thereafter). DNA from several engrafted sites in the mouse was extracted and PCR amplified before being sequenced on the Illumina HiSeq2500. Changes in pool complexity were analysed using MaGEcK software to determine which sgRNAs were significantly enriched or depleted. By far the most significantly enriched sgRNAs were those targeting NR3C1, the gene encoding the glucocorticoid receptor. In addition, two of the top five significantly depleted sgRNAs targeted the Plexins, PLXNA1 and PLXND1. Whilst PLXNA1 is expressed at low levels, PLXND1 is highly expressed and has been linked to dexamethasone resistance. Notably, the matched relapsed material from L707 was highly DEX resistant both in tissue culture and when transplanted into NSG mice. SNP 6.0 analysis revealed a 5q deletion in the relapse, spanning 5 genes including NR3C1. Whole genome sequencing showed this was comprised of 2 deletions both targeting NR3C1, with different breakpoints for each allele. The differential gene expression between the L707 presentation and relapse established that NR3C1 was the most significant of all the genes lost at relapse, based on gene set enrichment analysis (GSEA). This contrasts with many ALL cases, where one of the downstream effectors of apoptosis is lost as opposed to NR3C1. Growth of the relapse material in vivo and in vitro was slower than the presentation in a competitive situation, but with DEX treatment the relapse phenotype began to emerge with a small percentage of cells showing a heterozygous deletion of NR3C1. These combined data strongly suggest that the NR3C1 deletion is the main driver of DEX resistance in the L707 relapse. Moreover, it proves that our in vivo CRISPR screen predicted the leukaemic relapse. These results confirm NR3C1 deletion as a driver in glucocorticoid resistance and demonstrate the power of in vivo CRISPR screens to predict mechanisms of gain of drug resistance and subsequent relapse. The parallels that can be drawn between the relapse and the CRISPR screen are striking, giving the indication that the progression from presentation to relapse may follow the same path in a patient derived xenograft setting as it did in the patient. Disclosures No relevant conflicts of interest to declare.

CD138 Regulates Myeloma Cell Survival, Proliferation, BM Engraftment and Tumor Organization

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2974-2974
Author(s):  
David R Fooksman ◽  
Amitabha Mazumder ◽  
Mark McCarron
Keyword(s):  
Plasma Cells ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Early Stage ◽  
Serum Starvation ◽  
High Dose ◽  
Rapid Reduction ◽  
Myeloma Cells

Abstract Multiple myeloma is the 2nd most common blood cancer in adults with a median survival time of 5 years despite high-dose chemotherapy and bone marrow transplantation interventions. Syndecan-1 or CD138, is a heparan-sulfate coated glycoprotein, which is highly expressed on the surface of plasma cells and myeloma cells, important for adhesion and accumulating survival signals. Expression of CD138 is heterogeneous in myeloma tumors, in vivo and in vitro leading some to speculate it may distinguish stem-like subpopulations. While this role is highly disputed, we investigated the effect of CD138 expression on tumor pathology in vivo. To characterize CD138neg and CD138high subpopulations, we used GFP+ Vk*myc myeloma model from Leif Bergsagel, which develops myeloma tumors in BM and spleen of C57Bl/6 mice. We found CD138high populations were more proliferative in vivo based on EdU incorporation experiments. We transferred equal numbers of sorted subpopulations into hosts and found that CD138high cells generated larger tumors in the BM than CD138neg cells after 12 weeks. Analysis of these tumor-bearing mice revealed that all tumors contained both subpopulations, indicating that these two subsets are hierarchically equivalent. We find that in mice with small tumors, the majority of cells (80% or more) are CD138high cells, while in large tumors, the level drops (to 30-50% of tumor) with higher composition of CD138neg cells. We also find lower CD138 levels on myeloma cells found in the blood compared to BM. Using intravital two-photon time-lapse imaging in the tibial BM, we find that tumor cells from smaller, early stage tumors are physically arrested within the BM parenchyma, while in larger, more advanced tumors, myeloma cells are more motile and active. CD138neg cells were more apoptotic based on ex vivo Annexin V staining following serum starvation. Interestingly, serum starvation led to rapid reduction in CD138 surface expression. Taken together, we propose a model where CD138 expression regulates localization and survival in the BM niche, but is downregulated from the plasma membrane when tumor size outgrows the necessary resources, allowing myeloma cells to migrate and metastasize to distant new locations. Disclosures No relevant conflicts of interest to declare.

scholarly journals Dissecting targeted therapy resistance: Integrating models to quantify environment mediated drug resistance

2017 ◽  
Author(s):  
Noemi Picco ◽  
Erik Sahai ◽  
Philip K. Maini ◽  
Alexander R. A. Anderson
Keyword(s):  
Drug Resistance ◽  
Targeted Therapy ◽  
Targeted Therapies ◽  
Carrying Capacity ◽  
Conflicts Of Interest ◽  
Treatment Strategies ◽  
Model Systems ◽  
Significant Heterogeneity

AbstractDrug resistance is the single most important driver of cancer treatment failure for modern targeted therapies. This resistance may be due to the presence of dormant or aggressive tumor cell phenotypes or to context-driven protection. Non-malignant cells and other factors, constituting the microenvironment in which the tumor grows (the stroma), are now thought to play a crucial role in both therapeutic response and resistance. Specifically, the dialogue between the tumor and stroma has been shown to modulate the response to molecularly targeted therapies, through proliferative and survival signaling. The goal of this work is to investigate interactions between a growing tumor and its surrounding stroma in facilitating the emergence of drug resistance. We use mathematical modeling as a theoretical framework to bridge between experimental models and scales, with the aim of separating the intrinsic and extrinsic components of resistance in BRAF mutated melanoma. The model describes tumor-stroma dynamics both with and without treatment. Calibration of our model, through the integration of experimental data, revealed significant variation across animal replicates in either the intensity of stromal promotion or intrinsic tissue carrying capacity. Furthermore our study highlights the need to account for this variation in the design of treatment strategies. Major Findings. Through the integration of a simple mathematical model with in vitro and in vivo experimental growth dynamics of melanoma cell lines (both with and without drug), we were able to dissect the relative contributions of intrinsic versus environmental resistance. Our study revealed significant heterogeneity in vivo, indicating that there is a diversity of either stromal promotion or tumor carrying capacity under targeted therapy. We believe this variation may be one possible explanation for the heterogeneity observed across patients and within individual patients with multiple metastases. Therefore, quantifying this variation both within in vivo model systems and in individual patients could have a significant impact on the design of future treatment strategies that target both the tumor and stroma. Further, we present guidelines for building more effective and longer lasting therapeutic strategies utilizing our experimentally calibrated model. These strategies explicitly consider the protective nature of the stroma and utilize inhibitors that modulate it.PrecisQuantification of the environmental contribution to drug resistance reveals heterogeneity that significantly alters treatment dynamics that can be exploited for therapeutic gain.Financial SupportPicco and Anderson: US National Cancer Institute grant U01CA151924.Picco: UK Engineering and Physical Sciences Research Council (EPSRC grant number EP/G037280/1).Conflict of Interest DisclosureThe authors declare no potential conflicts of interest.

RFWD2 Induces Cellular Proliferation and Proteasome Inhibitor Resistance By Mediating p27 Ubiqutinaiton in Multiple Myeloma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3068-3068
Author(s):  
Ye Yang ◽  
Mengjie Guo ◽  
Chunyan Gu
Keyword(s):  
Multiple Myeloma ◽  
Cell Proliferation ◽  
Drug Resistance ◽  
Proteasome Inhibitor ◽  
Cellular Proliferation ◽  
Conflicts Of Interest ◽  
Scid Mice ◽  
Rpmi 8226

Purpose: In recent years, with the emergence of targeted proteasome inhibitors (PIs), the treatment of multiple myeloma (MM) has made great progress and significantly improves the survival rate of patients. However, MM remains an incurable disease, mainly due to the recurrence of drug resistance. The constitutive photomorphogenic 1 (RFWD2, also known as COP1), is closely related to the occurrence and development of tumors, but its role in MM is largely unknown. This study was aimed to explore the mechanism of RFWD2 on cell proliferation and resistance to proteasome inhibitor in MM. Experimental Design: Using gene expression profiling (GEP) samples, we verified the relation of RFWD2 to MM patients' survival and drug-resistance. The effect of RFWD2 on cell proliferation was confirmed by MTT and cell cycle analysis in RFWD2-overexpressed and RFWD2-knockdown MM cells. MTT and apoptosis experiments were performed to evaluate whether RFWD2 influenced the sensitivity of MM cells to several chemotherapy drugs. MM xenografts were established in immunodeficient NOD/SCID mice by injecting wild-type or RFWD2 over-expression MM cells with drug intervention. The mechanism of drug resistance was elucidated by analyzing the association of RFWD2 with E3 ligase of p27. Bortezomib-resistant RPMI 8226 cells were used to construct RFWD2 knockdown cells, which were injected into NOD/SCID mice to assess the effect of RFWD2 on bortezomib resistance in vivo. Results: RFWD2 expression was closely related to poor outcome, relapse and bortezomib resistance in MM patients' GEP cohorts. Elevated RFWD2 induced cell proliferation, while decreased RFWD2 inhibited cell proliferation and induced apoptosis in MM cells. RFWD2-overexpression MM cells resulted in PIs resistance, however, no chemotherapy resistance to adriamycin and dexamethasone was observed in vitro. In addition, overexpressing RFWD2 in MM cells led to bortezomib resistance rather than adriamycin resistance in myeloma xenograft mouse model. RFWD2 regulated the ubiquitination degradation of P27 by interacting with RCHY1 ubiquitin ligase. The knockdown of RFWD2 in bortezomib-resistant RPMI 8226 cells overcame bortezomib resistance in vivo. Conclusions: Our data demonstrate that elevated RFWD2 induces MM cell proliferation and resistance to PIs, but not to adriamycin and dexamethasone both in vitro and in vivo through mediating the ubiquitination of p27. Collectively, RFWD2 is a novel promising therapeutic target in MM. Disclosures No relevant conflicts of interest to declare.

scholarly journals Targeting CD47/TNFAIP8 by miR-155 overcomes drug resistance and inhibits tumor growth through induction of phagocytosis and apoptosis in multiple myeloma

Haematologica ◽  
2019 ◽  
Vol 105 (12) ◽  
pp. 2813-2823 ◽  
Author(s):  
Nasrin Rastgoo ◽  
Jian Wu ◽  
Mariah Liu ◽  
Maryam Pourabdollah ◽  
Eshetu G. Atenafu ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Drug Resistance ◽  
Tumor Growth ◽  
Myeloma Cell ◽  
Therapeutic Modality ◽  
Drug Resistant ◽  
Myeloma Cells ◽  
Advanced Stages

The mechanisms of drug resistance in multiple myeloma are poorly understood. Here we show that CD47, an integrin-associated receptor, is significantly upregulated in drug resistant myeloma cells in comparison with parental cells, and that high expression of CD47 detected by immunohistochemistry is associated with shorter progression free and overall survivals in multiple myeloma patients. We show that miR-155 is expressed at low levels in drug resistant myeloma cells and is a direct regulator of CD47 through its 3'UTR. Furthermore, low miR-155 levels are associated with advanced stages of disease. MiR-155 overexpression suppressed CD47 expression on myeloma cell surface, leading to induction of phagocytosis of myeloma cells by macrophages and inhibition of tumor growth. MiR-155 overexpression also re-sensitized drug-resistant myeloma cells to bortezomib leading to cell death through targeting TNFAIP8, a negative mediator of apoptosis in vitro and in vivo. Thus, miR-155 mimics may serve as a promising new therapeutic modality by promoting phagocytosis and inducing apoptosis in patients with refractory/relapsed multiple myeloma.

scholarly journals Elevated ANXA1 Expression Causes Glucocorticoid Resistance in Acute Lymphoblastic Leukemia Via Activating the Wnt/β-Catenin Signaling Pathway

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5198-5198
Author(s):  
Ping Liu ◽  
Dan Ma ◽  
Jishi Wang
Keyword(s):  
Western Blot ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Expression Profiles ◽  
Lymphoblastic Leukemia ◽  
Luciferase Assay ◽  
Protein Levels ◽  
Leukemia Relapse

Background: Acute lymphoblastic leukaemia (ALL) is one of the most common clonal malignant diseases in children, and it stems from unchecked proliferation of lymphoid progenitor cells. Glucocorticoids (GCs) such as prednisolone and dexamethasone are used as a chemotherapeutic drug in the treatment of ALL. GC-induced cell mortality is first mediated by the activation of glucocorticoid receptor (GR), followed by its translocation into the nucleus to activate or inhibit gene transcription. However, up to ~20% patients with leukemia relapse and become resistant to GCs. Therefore, a better understanding the molecular basis of chemoresistance in ALL would provide novel therapeutic opportunities for patients. Methods: By analyzing the published mRNA expression profiles (GSE5280; GSE94302) obtained from NCBI (https://www.ncbi.nlm.nih.gov/geo/), we found that higher expression of ANXA1 was significantly associated with decreased overall survival of ALL patients. We also examined the expression of ANXA1 at mRNA and protein levels in a variety of ALL cell lines by using qRT-PCR and western blot analyses. The mRNA and protein expression of ANXA1 in ALL cell lines and patients were determined using Real-time PCR and Western blot respectively. Functional assays, such as CCK-8, FACS, and Tunel assay used to determine the oncogenic role of ANXA1 in ALL progression. Furthermore, western blotting and luciferase assay were used to determine the mechanism of ANXA1 promotes chemoresistance in ALL cells. Results: The expression of ANXA1 was markedly upregulated in ALL cell lines and patients, and high ANXA1 expression was associated with relapsed/refractory ALL patients. ANXA1 overexpression confers glucocorticoids (GCs) resistance on ALL cells; however, down-regulated of ANXA1 sensitized ALL cell lines to GC both in vitro and in vivo. Additionally, ANXA1 upregulated the levels of FPRs by promoting Wnt/β-catenin signalling. Conclusions: Our findings provided evidence that ANXA1 is a potential therapeutic target for patients with ALL. Targeting ANXA1 signaling may be a promising strategy to enhance GC response during ALL chemo-resistance. Disclosures No relevant conflicts of interest to declare.

scholarly journals Mechanisms Regulating Myeloma Cell Responsiveness to Antisense Oligonucleotides Targeting IRF4

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 32-32
Author(s):  
Ryosuke Shirasaki ◽  
Esperanza M Algarín ◽  
Ricardo De Matos Simoes ◽  
Sondra L. Downey-Kopyscinski ◽  
Shizuka Yamano ◽  
...  
Keyword(s):  
High Risk ◽  
Cell Lines ◽  
Cell Response ◽  
Antisense Oligonucleotides ◽  
Research Funding ◽  
Mrna Levels ◽  
In Vivo Models ◽  
Genome Scale

Functional genomics studies from our group and others, including CRISPR screens, have documented that interferon regulatory factor 4 (IRF4) is a critical transcription factor (TF) for multiple myeloma (MM) cells in preclinical in vitro and in vivo models; and one of the top most pronounced and recurrent dependencies for MM cells vs. other neoplasias. IRF4 lacks known ligand-binding pocket(s) amenable to selective inhibition by small-molecule pharmacological agents. IRF4 is thus considered, as many TFs, as "undruggable". Recent progress in antisense oligonucleotides (ASOs) and their in vivo properties motivated us to systematically evaluate the response of MM cells to anti-IRF4 ASOs, to obtain direct insights into their potential therapeutic applications in MM and also as functional probes into IRF4 biology. We observed that a panel of anti-IRF4 ASOs (but not control ASO) exhibited dose- and time-dependent activity against genotypically diverse MM cell lines (sub-uM IC50s for most MM lines vs. &gt;10uM against non-MM cells lacking IRF4, e.g. HS5 bone marrow stromal cells [BMSCs]). Major in vitro anti-MM activity could be observed within 3-5 days of treatment; and was preceded by significant decrease in intracellular IRF4 protein levels. Importantly, 24-hour exposure to IRF4 ASO (followed by washout and subsequent culture in ASO-free media for up to 6 days) can induce similar anti-MM activity as continuous in vitro exposure over 6 days. Combinations of IRF4-ASO with several established (e.g. proteasome inhibitor, thalidomide derivative, glucocorticoids) or investigational (e.g. venetoclax) agents for MM result led to enhanced or even synergistic effects in a panel of 6 MM cell lines. For some of these MM cell lines, their in vitro response to IRF4 ASO had statistically significant attenuation in co-cultures with BMSCs: this effect was partially recapitulated in MM cell monocultures supplemented by conditioned media from BMSCs or (to a lesser extent) rhIL6 treatment. To obtain insights into cell-autonomous and nonautonomous mechanisms regulating MM cell responses to IRF4 ASOs, we examined the transcriptional profiles of MM.1S cells exposed to ASOs; and also performed genome-scale CRISPR studies to define genes whose editing (loss-of-function, LOF) or activation (gain-of-function, GOF) alter the MM cell response to the IRF4 ASOs. The transcriptional signature of MM cell treatment with IRF4 ASO (vs. control ASO) comprised distinct clusters of downregulated genes with preferential expression in normal or malignant plasma cells vs. other lineages; CRISPR-validated roles as dependencies for MM cells in vitro (preferentially essential for MM vs. non-MM tumors; or pan-essential); and/or proximity to large areas of chromatin accessibility (defined by H3K27Ac genome-wide ChIP-Seq or ATAC-Seq). Importantly, several genes downregulated by IRF4 ASO treatment are upregulated in MM cells cocultured with BMSCs, possibly explaining at least in part the impact of co-cultures on anti-MM activity of ASO. In our integrated genome-scale CRISPR studies (and validation of many genes with individual sgRNAs), MM cell response to IRF4 ASOs was attenuated by LOF of endonuclease RNase H1 (RNASEH1; which degrades the target RNA when it establishes intracellular hybrids with the ASOs); LOF of diverse genes involved in ASO endocytosis (e.g. IGF2R, PICALM, SH3GL1, RAB5C) or regulation of chondroitin/heparan sulfate (SLC35B2, B3GAT3, B4GALT7, ALG5 and ALG6); GOF of IRF4 itself (likely reflecting a "stoichiometric" effect, e.g. induction of higher IRF4 mRNA levels may require higher concentrations of ASO to maintain anti-MM effect); and GOF of exocytosis-related genes(e.g. CLU, QPCT). Importantly, LOF or GOF of individual genes typically associated with high-risk MM was not associated with decreased MM cell response to the IRF4 ASOs: therefore, retaining intracellular accumulation of IRF4 ASO and productive knockdown of IRF4 mRNA remains a primary driver of anti-MM activity of ASOs even in the context of biologically aggressive MM cells with "high-risk" features. Our results provide comprehensive integrated assessment of the molecular and functional landscapes associated with dysregulation of IRF4; have direct implications for our mechanistic understanding of the role of IRF4 in MM biology; and provide a framework for IRF4 targeting by ASOs or potentially other therapeutic approaches. Disclosures Downey-Kopyscinski: Rancho BioSciences, LLC: Current Employment. Luo:Ionis Pharmaceuticals, Inc.: Current Employment. Kim:Ionis Pharmaceuticals, Inc.: Current Employment. MacLeod:Ionis Pharmaceuticals, Inc.: Current Employment. Mitsiades:Arch Oncology: Research Funding; Sanofi: Research Funding; FIMECS: Consultancy, Honoraria; Karyopharm: Research Funding; Abbvie: Research Funding; Takeda: Other: employment of a relative; TEVA: Research Funding; Janssen/Johnson & Johnson: Research Funding; Fate Therapeutics: Consultancy, Honoraria; EMD Serono: Research Funding; Ionis Pharmaceuticals, Inc.: Consultancy, Honoraria.

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