Myeloma Cancer Stem Cells Research Articles

MicroRNA-138 promotes the progression of multiple myeloma through targeting paired PAX5

BackgroundMultiple myeloma cancer stem cells (MMSC) have been considered as the leading cause of multiple myeloma (MM) drug resistance and eventual relapse, microRNAs (miRNAs) collectively participate in the progression of MM. However, the pathogenesis of miR-138 in MMSC is still not fully understood. ObjectiveThe intention of this study was to investigate the mechanism and role of miR-138 in multiple myeloma. MethodBone marrow samples and peripheral blood from patients and normal controls were collected. Use Magnet-based Cancer Stem Cell Isolation Kit to separate and extract MMSC. Real-time quantitative PCR (RT-qPCR) was carried out to determine mRNA level. Western blot was applied to detect protein levels. MTT and flow cytometry were conducted to examine the proliferation and apoptosis of MMSC. Finally, dual-luciferase reporter gene assays were performed to confirm that paired box 5 (PAX5) is a direct target for miR-138. ResultsCompared with normal group, the expression of miR-138 in patients was significantly up-regulated, and the expression of miR-138 was in a negative correlation with PAX5. Additionally, downregulated miR-138 facilitated the apoptosis and inhibited the proliferation of MMSC in vitro and in vivo. Downregulated miR-138 moderated the expression of PAX5, Bcl-2, Bax, and Caspase-3. PAX5 was a direct target of miR-138. ConclusionTaken together, miR-138 plays a carcinogenic role in MM, and miR-138 adjusted the proliferation and apoptosis of MMSC by targeting PAX5. miR-138 has the probability of becoming a new medicinal target for the treatment of MM.

Cancer Stem Cells as a Prognostic Biomarker and Therapeutic Target Using Curcumin/ Piperine Extract for Multiple Myeloma.

Multiple myeloma (MM) is a hematological bone marrow malignancy that can be treated but is usually fatal. Medication resistance is the major cause of relapses due to cancer stem cells (CSCs). As a result, this study aimed to identify multiple myeloma cancer stem cells (MMCSCs) in the bone marrow of twelve MM patients with pathological complete response (pCR) after chemotherapy and to investigate the potential effect of Curcumin/Piperine (C/P) extract as an anti-MMCSCs treatment in twenty newly diagnosed patients. This study included twenty bone marrow (BM) samples from newly diagnosed MM patients and twelve BM samples from pCR patients after a year of treatment. The MTT test was performed to assess the treatment's effective dosage. A flow cytometer was used to identify MMCSCs, cell cycle profile, extract's apoptotic activity, and proliferation marker in the selected samples. Also, a colony formation test and stemness protein were investigated. In newly diagnosed MM patients, the C/P extract suppressed MMCSCs by 64.71% for CD138-/CD19- and 38.31% for CD38++. In MM patients' samples obtained after one year of treatment, the MMCSCs inhibition percentage reached 44.71% (P < 0.008) for CD138-/CD19- and 36.94% (P < 0.221) for CD38++. According to cell cycle analyses, the number of cells treated with C/P extract was significantly reduced in the S and G0/G1 phases (87.38%: 35.15%, and 4.83%: 2.17% respectively), with a rapid increase in the G2/M phases (1.1%: 2.2%.). MMCSCs apoptosis was identified using a flow cytometer and Annexin-V. Multiple myeloma stem cell (MMCSC) proliferation was inhibited. Clonogenicity was suppressed by 60%, and stemness protein expression was reduced by 70%. MMCSCs in the bone marrow of MM-pCR patients can be utilized as a prognostic tool to predict recurrent multiple myeloma incidence. Also, the therapeutic potential of C/P extract as a prospective anti-MM drug targeting MMCSCs.

Rs7853346 Polymorphism in lncRNA-PTENP1 and rs1799864 Polymorphism in CCR2 are Associated with Radiotherapy-Induced Cognitive Impairment in Subjects with Glioma Via Regulating PTENP1/miR-19b/CCR2 Signaling Pathway.

LncRNA-PTENP1 was reported to promote multiple myeloma cancer stem cell proliferation, and the G allele of rs7853346 polymorphism in lncRNA-PTENP1 was demonstrated to enhance the effect of lncRNA-PTENP1. In this study, we aimed to study the potential effect of lncRNA-PTENP1 and CCR2 mRNA polymorphisms on cognitive impairment in glioma patients. In this study, 279 glioma patients were recruited and grouped according to their genotypes of rs7853346 in PTENP1 and rs1799864 in CCR1. Pathogenic parameters were collected from patients before radiotherapy (month 0) or at month 1 and month 3 after radiotherapy to study the effect of rs7853346 and rs1799864 on cognitive impairment. Sequence analysis, luciferase assay, real-time PCR, and Western blot were performed to study the regulatory relationships between lncRNA-PTENP1, miR-18b, and CCR2. The glioma patient groups exhibited no significant differences concerning basic characteristics. However, the CG&GG/GG genotype alleviated radiotherapy-induced cognitive impairment by exhibiting the highest MMSE among the four groups. On the contrary, parameters including the severity of depression, bladder control, global health status, itchy skin, and weakness of legs all showed no difference among different patient groups at month 0, month 1, and month 3. Also, a long-term positive effect of CG&GG/GG genotype on role functioning and social functioning was also observed after radiotherapy. Compared with patients carrying the CC genotype of rs7853346, the expression of lncRNA-PTENP1 was reduced while the miR-19b level was elevated in patients carrying the CG&GG genotypes of rs7853346. Moreover, the expression of CCR2 mRNA was the highest in the CC/GA&AA group and the lowest in the CG&GG/GG group. Subsequent sequence analysis and luciferase assay indicated that miR-19b could bind to lncRNA-PTENP1 and 3'UTR of CCR2 mRNA, and the knockdown of lncRNA-PTENP1 led to evident up-regulation of miR-19b and down-regulation of CCR2 mRNA/protein in a cellular model, thus verifying the presence of the lncRNA-PTENP1/miR-19b/CCR2 mRNA signaling pathway. In conclusion, by studying the changes in the key parameters of glioma patients who were subjected to radiotherapy, we concluded that the rs7853346 polymorphism in lncRNA-PTENP1 and the rs1799864 polymorphism in CCR2 could independently affect cognitive impairment, while a more significant combined effect on cognitive impairment was exerted in glioma patients via the signaling pathway of PTENP1/miR-19b/CCR2.

CD138- multiple myeloma cells express high level of CHK1 which correlated to overall survival in MM patient.

Multiple myeloma (MM) is a disease in which abnormal plasma cells proliferate and secrete monoclonal immunoglobulin in the bone marrow. The main characteristic of plasma cells is the expression of the cell surface antigen syndecan-1 (CD138). However, the expression of CD138 is limited to terminally differentiated plasma cells during B cell development. A small subpopulation (2~5%) of human MM cells that lack CD138 expression has been shown to possess enormous proliferation potential in vitro experiment and in animal models, and they also can differentiate into CD138+ plasma cells. Thus, this small subset of MM cells was regarded as myeloma cancer stem cell (MCSC). However, its characteristics associated with the pathogenesis of MM remain unclear. In this study, we analyzed the gene expression data of CD138 cell lines downloaded from Gene Expression Omnibus (GEO) database. Limma package in RStudio was used to identify differentially expressed genes (DEGs). Genes enrichment and protein-protein interaction (PPI) network analysis were performed on DAVID and STRING databases. Furthermore, overall survival (OS) analysis in MM patient was utilized to screen out the hub-genes closely associate with the MM pathogenesis process. Hub-genes expression validation and receiver operating characteristic curve (ROC) analysis was performed in different stages of plasma cell disorder diseases. Finally, we verified these findings in MM patient samples. Through integrated bioinformatics analysis of MM CD138- and CD138+ cell lines, we found that CDC7, CDK1, and CHK1 are highly expressed in CD138- MM cells. These genes are crucial in the G2/M phase of the cell cycle pathway, which is closely related to the malignant proliferation in various tumor cells. Of note, we found that patients with high expression of CDC7, CDK1, and CHK1 had shorter overall survival time. The expression of CHK1 was significantly increased in MM cells compared with normal plasma cell (NPC) and MGUS. More importantly, we further clarified that the expression of CHK1 in release/refraction MM (R/R MM) has obviously increased compared with new diagnosed MM (ND MM).

G allele of rs7853346 polymorphism in PTENP1 enhances the proliferation of multiple myeloma cancer stem cells by promoting the expression of PTENP1 and its downstream signaling molecules.

Noncoding RNAs, including microRNAs (miRNAs) and long non-coding RNAs (lncRNAs), are shown to be associated with the pathogenesis of various diseases, such as multiple myeloma (MM). Therefore, the aim of this study was to explore the role of rs7853346 polymorphism in PTENP1, and its downstream signaling molecules, in the pathology of MM. Forty-three multiple myeloma patients and 35 healthy subjects were recruited and divided into CC, CG, and GG groups according to their genotypes of rs7853346 polymorphism in PTENP1. Real-time polymerase chain reaction (PCR), Western-blot analyses and immunohistochemistry assays were utilized to compare the expression of PTENP1, miR-19b, and TSC1 between different groups. In addition, the relationship between PTENP1, miR-19b, and TSC1, as well as the role of PTENP1 in MM, was explored. Higher levels of PTENP1 and TSC1 mesenger RNA (mRNA) were observed in the MM group, along with a lower level of miR-19b. Moreover, the protein level of TSC1 in the MM group was evidently upregulated compared to that in the negative control (NC) group. Meanwhile, compared to the CG and GG groups, the CC group showed higher levels of PTENP1 and TSC1 mRNA, as well as a lower level of miR-19b. According to the results of real-time PCR, a negative correlation with a correlation coefficient of -0.05 was established between PTENP1 and miR-19b expression. Similarly, a negative correlation with a correlation coefficient of -0.05 was also established between miR-19b and TSC1 expression. In addition, the transfection of pcDNA-PTENP1 or anti-miR-19b into cells significantly suppressed miR-19b expression but obviously increased TSC1 expression. The G allele of rs7853346 polymorphism enhances the proliferation of MM cancer stem cells by promoting the expression of PTENP1 as well as its downstream signaling molecules. Therefore, rs7853346 may become a novel biomarker for the diagnosis and treatment of MM.

Activated Leukocyte Cell Adhesion Molecule (ALCAM) Regulate Myeloma Cancer Stem Cell Mediated Myelomagenesis and Chemoresistance

IntroductionMultiple myeloma (MM) is an incurable plasma cell cancer. People introduced the concept of cancer stem cells (CSCs) in MM, known as MM-CSCs, to characterize a small population of malignant cells with distinguishable activities of self-renewal and differentiation into regular MM cells. In this study, we showed that activated leukocyte cell adhesion molecule (ALCAM) interacted with epidermal growth factor receptor (EGFR). ALCAM-EGFR axis regulated MM-CSC mediated myelomagenesis and chemoresistance.MethodsMM-CSCs in MM cell lines and primary MM cells were identified by Hoechst staining or ALDEFLUOR dye, followed by flow cytometry analyses. MM-CSC clonalization activity was examined by soft agar colony formation assay. The protein-protein interaction was identified by co-immunoprecipitation, proximity ligation assay, and proteins' structure simulation. MM-CSC mediated myelomagenesis in vivo was examined in human MM xenograft mouse model and murine adoptive MM model. MM-CSC mediated chemoresistance in vivo was examined using 5T MM model. The clinical significance of ALCAM expression in MM was analyzed using patients' gene expression profiles.ResultsBoth MM cell lines and primary MM cells expressed ALCAM. When cultured with bone marrow stromal cells (BMSCs) or in BMSC-conditioned medium, ALCAM knocked down (AL-KD) MM cells exhibited increased colony formation activity. Hoechst staining showed that under such culture conditions, AL-KD MM cells had increased stemness side population (SP), which was MM-CSC. Depletion of MM-CSC reduced MM colony formation activity. In human MM xenograft model, subcutaneous co-injection of AL-KD MM cells and BMSCs had large amount of MM-CSC and fast tumorigenesis. In murine adoptive MM model, which had tumor derived from viral transformed plasma cells with mandatory expression of MYC, KRAS genes and a shRNA sequence, ALCAM shRNA expression resulted significant more rapid tumor development than non-target shRNA.Mechanistic studies showed that ALCAM and EGFR co-immunoprecipitated in MM cells. Proximity ligation assay showed interaction of ALCAM and EGFR in primary MM cells. Immunofluorescent staining showed that ALCAM colocalized with unphosphorylated EGFR in MM. Epidermal growth factor (EGF), the canonical ligand of EGFR, stimulated EGFR phosphorylation and abolished ALCAM-EGFR colocalization. Protein structure simulation suggested that EGF and ALCAM mutual exclusively bound to the same region of EGFR. EGF addition stimulated MM-CSC in cultured MM. Inhibitors of EGFR, ERK, or hedgehog pathway repressed MM-CSC. EGFR activation led to hedgehog pathway activation via RAS/MEK/ERK cascade, while ALCAM inhibited such pro-MM-CSC cell signaling by binding to EGFR.Chemotherapeutics, such as bortezomib and melphalan, induced MM apoptosis, but also stimulated MM-CSCs, which were resistant to chemotherapeutics. Although CTR-KD and AL-KD MM had similar death rate after the drug treatment, AL-KD MM cells had more MM-CSC generated. MM-CSCs derived in AL-KD MM cells had more active cell proliferation even in the presence of drugs. Further study showed that chemotherapy stimulated EGFR expression and repressed ALCAM expression, therefore switched the balance of ALCAM-EGFR axis to pro-MM-CSC direction. EGFR inhibitors, such as gefitinib, did not have anti-MM activity, but repressed MM-CSCs. In 5T MM model, Gefitinib/melphalan combination therapy resulted prolonged remission period, and significant better mouse survival.We identified MM-CSC in patients BM. ALCAM expression correlated with MM overall survival (OS): ALCAMhigh patients had better OS. In ALCAM-EGFR axis, only ALCAM expression, but not EGFR expression, correlated with MM OS. ALCAM expression varied in different patients, while EGFR expression was more even. Therefore we believe in ALCAM-EGFR axis, it was ALCAM, but not the EGFR, that played the regulatory role. Furthermore, pathway enrichment analysis showed that MM-CSC, identified by Hoechst staining, and MM minimum residual disease (MRD) shared similarities in signaling pathways, as well as pattern of ALCAM expressions.Conclusion and DiscussionALCAM-EGFR axis regulates MM-CSC mediated myelomagenesis and chemoresistance. EGFR inhibitors repress chemoresistant MM-CSC. Combination therapy with conventional chemotherapeutics and an EGFR inhibitor might be promising in MM treatment. DisclosuresNo relevant conflicts of interest to declare.

Anti-inflammatory and antiproliferative diterpenoids from Plectranthus scutellarioides

Chemical investigation of the dichloromethane extract of the aerial parts of Plectranthus scutellarioides led to the isolation and characterization of 10 diterpenoids with an abietane skeleton and one cembrane-type diterpenoid. Among them, six have not yet been described in the literature. Their structures were established by 1D and 2D NMR, UV and IR spectroscopy, and HRESIMS. The relative configuration was determined by Gauge-Independent Atomic Orbital NMR chemical shift calculations supported by the advanced statistical method DP4 plus and further confirmed by electronic circular dichroism. The isolated constituents were evaluated for their in vitro NF-κB inhibitory activity, as well as for their cytotoxic effects in human multiple myeloma cancer stem cells and RPMI 8226 tumor cell line. Coleon O, coleon G, lanugone K and 6-acetylfredericone B showed the highest inhibitory effect against NF-κB, displaying IC50 of 11.2, 11.0, 4.5 and 9.7 μM, respectively. Coleon O exhibited also a significant activity towards human multiple myeloma cancer stem cells and RPMI 8226 cells with IC50 of 9.2 and 8.4 μM, respectively.

Induction of multiple myeloma cancer stem cell apoptosis using conjugated anti-ABCG2 antibody with epirubicin-loaded microbubbles

BackgroundMultiple myeloma (MM) currently remains largely incurable. Cancer stem cells (CSCs) are believed to be responsible for drug resistance and eventual relapse. In this study, we exploited a novel agent to evaluate its inhibitory effect on MM CSCs.MethodsEpirubicin (EPI)-loaded lipid microbubbles (MBs) conjugated with anti-ABCG2 monoclonal antibody (EPI-MBs + mAb) were developed and their effect on MM 138−CD34− CSCs isolated from human MM RPMI 8226 cell line plus ultrasound exposure in vitro and in vivo in a nonobese diabetic/severe combined immunodeficient mouse model were assessed.ResultsEPI-MBs + mAb combined with ultrasound led to a significant decrease in the clone formation ability and the mitochondrial membrane potential along with an increase in MM CSC apoptosis. Moreover, treatment with EPI-MBs + mAb with ultrasound exposure remarkably inhibited the growth of MM CSC-derived tumors in xenograft nonobese diabetic/severe combined immunodeficient mice compared with a single agent or EPI-MBs + mAb without ultrasound exposure. The inhibitive efficacy was also correlated with an increased expression of caspase-3, Bax, and TUNEL and decreased expressions of PCNA, Bcl-2, and CD31.ConclusionsOur findings reveal that the EPI-MBs + mAb combined with therapeutic ultrasound may confer an effective approach for treatment of MM by induction of an apoptotic pathway in MM CSCs.

Bioactive metabolites from the leaves of Withania adpressa

Context: Withania (Solanaceae) species are known to be a rich source of withanolides, which have shown several biological properties.Objective: To identify the compounds responsible for Withania adpressa Coss. antioxidant activity and further test them for their NF-κB inhibition and antiproliferative activity in multiple myeloma cells.Materials and methods: Compounds were obtained from the EtOAc extract of W. adpressa leaves. Structure elucidation was carried out mainly by 1D- and 2D-NMR, and mass spectrometry. Isolated compounds were tested in a dose-response for their in vitro NF-κB inhibition and antiproliferative activity in multiple myeloma cells after 5 and 72 h treatment, respectively.Results: The fractionation resulted in the isolation of a new glycowithanolide named wadpressine (5) together with withanolide F, withaferin A, coagulin L, and nicotiflorin. The latter showed a moderate ability to scavenge free radicals in DPPH (IC50 = 35.3 µM) and NO (IC50 = 41.3 µM) assays. Withanolide F and withaferin A exhibited low µM antiproliferative activity against both multiple myeloma cancer stem cells and RPMI 8226 cells. Furthermore, they inhibited NF-κB activity with IC50 values of 1.2 and 0.047 µM, respectively. The other compounds showed a moderate inhibition of cell proliferation in RPMI 8226 cells, but were inactive against cancer stem cells and did not inhibit NF-κB activity.Discussion and conclusions: One new glycowithanolide and four known compounds were isolated. Biological evaluation data gave further insight on the antitumor potential of withanolides for refractory cancers.

Targeting Multiple Myeloma Cancer Stem Cells with Natural Products - Lessons from Other Hematological Malignancies.

Multiple myeloma is characterized by the accumulation of malignant plasma cells in the bone marrow. Multiple myeloma is the second most frequently diagnosed hematological malignancy, predominantly affecting the elderly. Despite recent advances in the development of novel therapies, multiple myeloma remains an incurable malignancy where the majority of patients relapse, develop resistance, and eventually die from the disease. This has been attributed to the fact that conventional therapy currently in use targets mainly the bulk of tumor cells, but not the tumor-initiating cancer stem cells. Cancer stem cells are a highly resistant subpopulation of cells believed to be responsible for the initiation, progression, metastasis, and relapse of cancer. Enormous efforts have been invested in the characterization of cancer stem cells. These efforts led to the characterization of key cellular signaling pathways responsible for conferring stem cell characteristics including self-renewal, differentiation, migratory, survival, and intracellular detoxification capabilities. Targeting these protective mechanisms offers a valuable strategy that may help combat a major driving force behind cancers. The use of natural products offers a promising therapeutic approach for targeting cancer stem cells. In this review, recent advances achieved in the characterization of cancer stem cells derived from hematological malignancies, with a particular focus on multiple myeloma, are discussed and major natural products that target cancer stem cells are presented. As natural products remain an essential source of novel chemical structures and medicinal leads, the exploitation of this immense reservoir is used to draw lessons in targeting multiple myeloma-cancer stem cells.

PEGylated long-circulating liposomes deliver homoharringtonine to suppress multiple myeloma cancer stem cells.

The goal of this investigation was to evaluate the inhibiting effect of high proportion polyethyleneglycol of long-circulating homoharringtonine liposomes on RPMI8226 multiple myeloma cancer stem cells. The CD138-CD34- multiple myeloma cancer stem cells isolated from RPMI8226 cell line using magnetic activated cell sorting system were, respectively, incubated with the optimized formulation of polyethyleneglycol of long-circulating homoharringtonine liposomes and the homoharringtonine invitro, and the multiple myeloma cancer stem cell proliferation, colony formation, and cell cycle were analyzed. The inhibition of the multiple myeloma CD138-CD34- cancer stem cell growth was investigated in non-obese-diabetic/severe-combined-immunodeficiency mice that were implanted with multiple myeloma RPMI 8226 cancer stem cells and treated with the LCL-HHT-H-PEG. The results showed that the polyethyleneglycol of long-circulating homoharringtonine liposomes significantly inhibited MM cancer stem cell proliferation, colony formation, and induced cancer stem cell apoptosis invitro as well as MM cancer stem cell growth in non-obese-diabetic/severe-combined-immunodeficiency mice compared with the homoharringtonine. In addition, the mouse bone mineral density and the red blood cell count were significantly increased in polyethyleneglycol of long-circulating homoharringtonine liposomes group. In conclusion, the data demonstrated that the developed polyethyleneglycol of long-circulating homoharringtonine liposomes formulation may serve as an efficient therapeutic drug for suppressing CD138-CD34- multiple myeloma cancer stem cell growth by inducing cancer stem cell apoptosis in non-obese-diabetic/severe-combined-immunodeficiency mouse model. Impact statement Multiple myeloma (MM) remains largely incurable until now. One of the main reasons is that there are cancer stem cells (CSCs) in MM, which are responsible for MM's drug resistance and relapse. In this study, we wanted to extend our previous investigation22 that whether we developed the LCL-HHT-H-PEG formulation have an inhibitory effect on MM CD138-CD34-CSCs in MM CSC engrafted NOD/SCID mouse model. Our data from the present study have demonstrated the therapeutic effect of LCL-HHT-H-PEG on MM-bearing mouse model. The study represents the first attempt to demonstrate that the LCL-HHT-H-PEG formulation is available for treatment MM patients in clinic. Therefore, this finding is important and deserves publication in Experimental Biology and Medicine.

Withaferin A induces cell death and differentiation in multiple myeloma cancer stem cells.

Multiple myeloma (MM) remains an incurable malignancy despite the development of novel therapeutics. This is believed to be due to a subset of rare chemotherapy-resistant cancer stem cells (CSCs). Differentiation therapy represents one strategy aimed at reducing the stemness of CSCs. The anticancer effect of withaferin A (WFA) was studied in MM-CSCs and RPMI 8226 MM tumoral plasma cells (RPMIs). WFA exhibited growth inhibitory effects in both MM-CSCs and RPMIs, with IC50 values of 649 and 224 nM, respectively. WFA also induced a G2 cell cycle arrest, as well as cell death and apoptosis. Although, WFA did not exhibit a direct anti-migratory effect, a remarkable morphological change was observed in MM-CSCs in response to WFA treatment. Using qPCR gene expression analyses, WFA caused a reduction in stemness markers, and a promotion of differentiation markers in MM-CSCs. These results warrant further investigation of WFA in relevant MM animal models.

Multiple Myeloma Cancer Stem Cells: Immunophenotypic and Functional Characterization, Gene Expression Profiling and Therapeutic Targets

Background: Despite the increase in patients' survival over the last years, multiple myeloma (MM) remains incurable, being persistence of cancer stem cells (CSCs) a probable cause of drug resistance and disease relapse. It is possible to isolate these cells using surface antigen expression pattern (CD19+/CD34+/CD138-) and the activity of an enzyme from aldehyde dehydrogenase (ALDH) family (Boucher et al., 2012). We believe that using CD19 as potential marker of MM-CSCs makes CAR-T cell therapy against CD19 an option to eradicate residual MM disease.Aims: To isolate and characterize immunophenotypically, functionally and by gene expression the MM-CSCs derived from bone marrow (BM) samples of newly-diagnosed MM patients, focusing on identification of possible therapeutic targets.Methods: BM aspirates were collected and CD138+ cells were separated by magnetic sorting. The remaining cells were submitted to sorting by flow cytometry on FACSAria II (Becton, Dickinson and Company, Franklin Lakes, NJ, USA), labeled with anti-CD19 Pacific Blue (Invitrogen, Carlsbad, CA, USA), anti-CD34 PE Cy7 and anti-CD138 APC (both from Becton, Dickinson and Company, Franklin Lakes, NJ, USA) antibodies, in addition to Aldefluor™ reagent (StemCell Technology, Vancouver, British Columbia, Canada). RNA was extracted and pre-amplified for PCR array analysis using the RT² Profiler™ PCR Array Human Cancer Stem Cells(Qiagen, Hilden, Germany) to assess the expression profile of 84 genes related to cancer stem cells, and the results were evaluated with the online software provided by the platform manufacturer.Results: MM-CSCs (CD34+/CD19+/CD138-/ALDH1+) were isolated by flow cytometry from MM samples and presented median of 1,748.5 events (ranging from 56 to 16,633, n = 16). For comparison purposes, CD138+ MM tumor cells were isolated and used as "control group" (median of events 72,904, ranging from 1,536 to 312,504, n = 15). RNA from 16 MM-CSC samples and 6 controls were analyzed by qPCR. Considering 2-ΔCt calculation (GAPDH as normalizer) and fold change of 2, 11 genes were considered differentially expressed in MM-CSCs when compared to tumor plasma cells (p<0.05) (4 overexpressed and 7 underexpressed), as calculated by Student’s T-test. Overexpression of CD38 in the control group confirms the origin of tumor plasma cells. Other genes overexpressed in tumor plasma cells include ITGA6 and PECAM1 (adhesion-related molecules); MERTK (signal transduction); BMI1; DKK1 (Wnt signaling pathway), and ID1 (cell migration and metastasis). Among the MM-CSCs overexpressed genes, ALDH1A1, ENG and ITGB1 are CSC markers and are related to stem cell functions; and WEE1 is a cell cycle-related gene which regulates G2-M checkpoint and promotes cell cycles arrest for further DNA repair.Discussion and Conclusions: Our preliminary data shows that cells isolated using Boucher et al. (2012) selected markers have MM-CSCs characteristics, validated by the expression of at least three genes related to CSCs: ALDH1A1, ENG and ITGB1. We also identified WEE1overexpression in MM-CSCs and this data is a new finding in MM studies. WEE1 overexpression has been described in several cancers, such as mantle cell lymphoma, colorectal cancer, breast cancer and glioblastoma. WEE1 inhibition by siRNAs or small molecule inhibitors AZD1775 (MK1775) and PD0407824, as shown in preclinical and clinical trials, impairs G2-M arrest and promotes premature entry in mitosis, leading to consequent cell death if DNA damage is irreparable (Mueller & Haas-Kogan, 2015). Therefore, these inhibitors could also be explored in MM treatment. Furthermore, we suggest that CAR-T cell therapy against CD19 might be and option to eradicate residual MM disease, since we confirmed that this antigen is present in MM-CSCs surface.Financial support: FAPESP 2010/17668-6 and FAPESP 2015/23983-5.Ethical approval: 0127/2014.Disclaimer: Oliveira MB and Dantonio PM equally contributed to this abstract. DisclosuresNo relevant conflicts of interest to declare.

Bruceantin inhibits multiple myeloma cancer stem cell proliferation

ABSTRACTMultiple myeloma (MM) continues to claim the lives of a majority of patients. MM cancer stem cells (CSCs) have been demonstrated to sustain tumor growth. Due to their ability to self-renew and to express detoxifying enzymes and efflux transporters, MM-CSCs are rendered highly resistant to conventional therapies. Therefore, managing MM-CSCs characteristics could have profound clinical implications. Bruceantin (BCT) is a natural product previously demonstrated to inhibit the growth of MM in RPMI 8226 cells-inoculated mouse xenograft models, and to cause regression in already established tumors. The objectives of the present study were to test the inhibitory effects of BCT on MM-CSCs growth derived from a human primary tumor, and to explore a mechanism of action underlying these effects. BCT exhibited potent antiproliferative activity in MM-CSCs starting at 25 nM. BCT induced cell cycle arrest, cell death and apoptosis in MM-CSCs as well as inhibited cell migration and angiogenesis in vitro. Using a qPCR screen, it was found that the gene expression of a number of Notch pathway members was altered. Pretreatment of MM-CSCs with the γ-secretase inhibitor RO4929097, a Notch pathway inhibitor, reversed BCT-induced effects on MM-CSCs proliferation. In this study, BCT was shown to be an effective agent in controlling the proliferation, viability and migration of MM-CSCs as well as angiogenesis in vitro. The effect on MM-CSCs proliferation may be mediated by the Notch pathway. These results warrant further investigation of BCT in a broader set of human-derived MM-CSCs and with in vivo models representative of MM.

Abstract 2232: Down-regulation of dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1A mediated by miR-1246 and miR-1290 in multiple myeloma cancer stem-like cells

Abstract Multiple Myeloma (MM) is a clonal plasma cell derived malignancy characterized with high relapse rate and fatality despite recent therapeutic advances. The existence of the rare population of CD138neg cancer stem-like cells had been identified and demonstrated to bear higher clonogenicity and self-renewal capacity when compared to the CD138pos major population. Besides, CD138neg MM cells are more resistant to common therapeutic agents treating MM including dexamethasone, lenalidomide and bortezomib. Therefore, targeting MM cancer-stem like cells could be critical to achieve a cure in MM. Previously, our lab had identified 2 microRNAs, miR-1246 and miR-1290 that are overexpressed in CD138neg MM cells in both cell lines and patient samples. Their target genes are being studied in the present study. CD138pos and CD138neg MM cells were isolated using MACS-immunomagnetic system and the purity of both CD138neg and CD138pos fraction were accessed by flow cytometry using PE anti-human CD138 antibody. The purity of each fraction was over 95% in 4 cell lines (U266, RPMI8226, OPM2 and KMS-12-PE) used. mRNA level of 7 putative targets (DYRK1A, KDM5A, NKRF, STK17A, PHLDA1, FOXA1, SCAI) chosen from TargetScan 6.2, miRanda or previous studies, which are related to tumor development in other cancers, of miR-1246 or miR-1290 were examined using RT-qPCR. Among the 7 putative targets, DYRK1A, KDM5A and NKRF were down-regulated in all four cell lines used. 3′UTR of the 3 down-regulated targets were cloned into pmirGLO Dual-Luciferase miRNA Target Expression Vector, which were co-transfected with miRNA-mimic into 293FT cells. Down-regulation of luciferase signal was observed in DYRK1A (p&amp;lt;0.05) and KDM5A (p&amp;lt;0.05). Mutagenesis of miR-1246 and miR-1290 binding sites of DYRK1A (but not in KDM5A) led to restoration of luciferase signal. Besides, mRNA and protein level of DYRK1A were also down-regulated 48 hours post-transfection of miRNA-mimics. The mRNA level of DYRK1A were also found to be significantly down-regulated in CD138neg cells when compared to CD138pos cells in 30 pairs of MM patient samples. Therefore, we conclude that DYRK1A should be a target of miR-1246 and miR-1290 in MM cells. Next, DYRK1A was knocked down by shRNA in U266. Preliminary results showed that down-regulation of DYRK1A reduced the percentage of apoptotic cells induced by treatment with bortezomib for 24 hours (p&amp;lt;0.05). Taken together, we had identified DYRK1A, which is down-regulated in MM cancer stem-like cells and regulated by miR-1246 and miR-1290. It may play a role in drug resistance in MM cancer stem-like cells. Our findings may provide insight on how MM cancer stem-like cells would escape from chemotherapy and facilitate the development of a novel therapeutic target in effective MM therapy. Citation Format: Terry Hei Yan Wong, Coty HY Cheung, Natalie Pui Ha Chan, Rosalina KL Ip, Chi-Keung Cheng, Kitty Ka Yan Ng, Margaret H.L. Ng. Down-regulation of dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1A mediated by miR-1246 and miR-1290 in multiple myeloma cancer stem-like cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2232. doi:10.1158/1538-7445.AM2015-2232

Inhibitory effect of epirubicin-loaded lipid microbubbles with conjugated anti-ABCG2 antibody combined with therapeutic ultrasound on multiple myeloma cancer stem cells

Ultrasound-targeted microbubble destruction (UTMD) technique is thought to improve the chemotherapeutic agent delivery from microbubbles (MBs) in tumor tissues and reduce the side effects in non-tumor tissues. Multiple myeloma (MM) is a bone marrow cancer and remains to be an incurable disease. In this study, we used the UTMD technique to investigate the inhibitory effect of our developed novel reagent on MM cancer stem cells (CD138−CD34−MM CSCs) that are MM cells with CD138−CD34− phenotypes, responsible for MM-initiating potential, drug resistance and eventual relapse. The preparatory steps of novel reagent was first epirubicin (EPI)-loaded in the lipid MBs that was consisted of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]-biotin, dipalmitoyl-phosphatidylglycerol and 25-NBD-cholesterol, then anti-ABCG2 monoclonal antibody (mAb) was conjugated onto the MB surface to form EPI-MBs+mAb. CD138−CD34−MM CSCs were isolated from human MM RPMI 8226 cell line by the magnetic associated cell sorting method. The results showed that the attenuated proliferation, migration and invasion ability, and increased apoptosis were observed when MM CSCs were incubated with a various agents. EPI-MBs+mAb combined with therapeutic ultrasound significantly promoted the MM CSC apoptosis compared with EPI, EPI-MBs alone or EPI-MBs+mAb without ultrasound exposure. These results suggest that the developed EPI-MBs+mAb combined with therapeutic ultrasound remarkably induced MM CSC apoptosis in vitro.

Abstract 4049: Stromal Wnt/β-catenin antagonism with DKK1 promotes clonal expansion of multiple myeloma is identified using hyaluronic acid based 3D hydrogel

Abstract Multiple myeloma (MM), an incurable cancer with 3- to 5-year survival is characterized by monoclonal immunoglobulin (Ig), lytic bone lesions and monoclonal plasma cells (PCs) in the bone marrow (BM). Because clonal expansion of primary MM cells outside their BM microenvironment has been unsuccessful, most pre-clinical studies have used MM cell lines derived from leukemic-phase cells that have escaped BM dependence. Therefore it is likely that BM niches maintain MM cancer stem cells (MM-CSCs) in a quiescent, drug-resistant state. To date, pre-clinical models do not take into account adhesion-mediated drug resistance and none allows for testing of drug efficacy on MM-CSC populations. Mouse models of MM are inadequate for pre-clinical use because they cannot faithfully recapitulate human disease. Recently, a stromal spheroid co-culture model and various scaffolds have been developed to recreate the 3D environment of the BM. To adequately investigate underlying mechanisms of B-cell development and the stromal influence on MM pathogenesis, there is a great need for a 3D cell culture system. In the present study, we used hyaluronic acid based 3D hydrogel for surface seeding and encapsulation of human multiple myeloma (BMMCs) and the bone marrow stromal cells (BMSCs) to investigate MM clonal migration and the new phenotype exhibiting cellular changes associated with Wnt/βcatenin signaling. For the first time we report, (a) hyaluronic acid HA-based 3D hydrogels with difference in the matrix composition and stiffness provide a suitable substratum for migration and clonal expansion of human primary BMMCs; (b) Homogenous encapsulation of 5x105 BMMCs showed development of small colonies identified as multiple thin, flat cells emanating from a central cluster of rounded cells as colony forming units (CFUs). We observed an increase in CFUs in hydrogel with medium stiffness of Me-HA at 14 days (ranging from 16 to 17%, p&amp;lt;0.05) and at 21 days (ranging from 26 to 27%, p&amp;lt;0.01) in contrast to that in 2D or in a commercial matrigel; (c) immunofluorescence detection of cytoplasmic and nuclear expression of β-catenin showed a higher expression (&amp;gt;4 fold) in the 3D encapsulated differentiating cells compared to that in the non-differentiating single cells; (d) our findings using ELISA assays for sDKK1 showed a gradual increase in DKK1 with a maximum at 21 days (p&amp;lt;0.001), suggesting the possible influence of stromal BMSCs on DKK1 production in contrast to earlier reports of DKK1 inhibiting Wnt signaling. Our findings support the use of 3D (ex vivo system) hydrogel for MM to dissect the Wnt-β-catenin/DKK1 signaling activities and further to screening for drug targets in bone-related human multiple myeloma. Citation Format: Bhagavathi A. Narayanan, Bin Duan, Jonathan T. Butcher, Amitabha Mazumder, Narayanan K. Narayanan. Stromal Wnt/β-catenin antagonism with DKK1 promotes clonal expansion of multiple myeloma is identified using hyaluronic acid based 3D hydrogel. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4049. doi:10.1158/1538-7445.AM2014-4049

Activation of liver X receptors inhibits hedgehog signaling, clonogenic growth, and self-renewal in multiple myeloma.

The Hedgehog (Hh) signaling pathway is aberrantly activated in a wide variety of human cancers, and recent clinical studies have demonstrated that pathway inhibitors are effective in advanced basal cell carcinoma (BCC). The majority of these agents have been designed to target SMOOTHENED (SMO), a transmembrane regulator of Hh signaling, but subsequent mutations in SMO have been found to generate drug resistance. In other cancers, oncogenic events that bypass SMO may activate canonical Hh signaling, and SMO antagonists have not demonstrated significant activity in several diseases. Therefore, alternative strategies targeting the Hh pathway downstream of SMO may have clinical utility. Liver X receptors (LXR) regulate cholesterol and fatty acid homeostasis, and LXR activation can inhibit the Hh pathway in normal mouse embryonic fibroblasts. We examined the effects of LXR activation on Hh signaling in human multiple myeloma cells and found that LXR agonists inhibited Hh pathway activity and clonogenic tumor growth in vitro. LXR activation also inhibited putative multiple myeloma cancer stem cells in vivo leading to the loss of tumor initiating and self-renewal potential. Finally, Hh signaling was inhibited downstream of SMO, suggesting that LXR agonists may represent a novel strategy to target pathogenic Hh signaling as well as treat multiple myeloma.

737: ICP-133 controls in vitro cell proliferation of multiple myeloma cancer stem cells

737: ICP-133 controls in vitro cell proliferation of multiple myeloma cancer stem cells

Phenotypic, genomic and functional characterization reveals no differences between CD138++ and CD138low subpopulations in multiple myeloma cell lines.

Despite recent advances in the treatment of multiple myeloma (MM), it remains an incurable disease potentially due to the presence of resistant myeloma cancer stem cells (MM-CSC). Although the presence of clonogenic cells in MM was described three decades ago, the phenotype of MM-CSC is still controversial, especially with respect to the expression of syndecan-1 (CD138). Here, we demonstrate the presence of two subpopulations - CD138++ (95–99%) and CD138low (1–5%) - in eight MM cell lines. To find out possible stem-cell-like features, we have phenotypically, genomic and functionally characterized the two subpopulations. Our results show that the minor CD138low subpopulation is morphologically identical to the CD138++ fraction and does not represent a more immature B-cell compartment (with lack of CD19, CD20 and CD27 expression). Moreover, both subpopulations have similar gene expression and genomic profiles. Importantly, both CD138++ and CD138low subpopulations have similar sensitivity to bortezomib, melphalan and doxorubicin. Finally, serial engraftment in CB17-SCID mice shows that CD138++ as well as CD138low cells have self-renewal potential and they are phenotypically interconvertible. Overall, our results differ from previously published data in MM cell lines which attribute a B-cell phenotype to MM-CSC. Future characterization of clonal plasma cell subpopulations in MM patients' samples will guarantee the discovery of more reliable markers able to discriminate true clonogenic myeloma cells.