scholarly journals The CXCL12gamma chemokine immobilized by heparan sulfate on stromal niche cells controls adhesion and mediates drug resistance in multiple myeloma

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Zemin Ren ◽  
Hildo Lantermans ◽  
Annemieke Kuil ◽  
Willem Kraan ◽  
Fernando Arenzana-Seisdedos ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Drug Resistance ◽  
Heparan Sulfate ◽  
Stromal Cells ◽  
Functional Roles ◽  
Bm Niche ◽  
Human Bmscs ◽  
Chemokine Cxcl12 ◽  
Mrna And Protein Expression ◽  
Membrane Bound

Abstract Background The survival and proliferation of multiple myeloma (MM) cells in the bone marrow (BM) critically depend on interaction with stromal cells expressing the chemokine CXCL12. CXCL12 regulates the homing to the BM niche by mediating the transendothelial migration and adhesion/retention of the MM cells. The gamma isoform of CXCL12 (CXCL12γ) has been reported to be highly expressed in mouse BM and to show enhanced biological activity compared to the ‘common’ CXCL12α isoform, mediated by its unique extended C-terminal domain, which binds heparan sulfate proteoglycans (HSPGs) with an extraordinary high affinity. Here, we investigated the expression of CXCL12γ in human BM and studied its functional role in the interaction of MM cells with BM stromal cells (BMSCs). Methods We assessed CXCL12γ mRNA and protein expression by human BMSCs using qPCR, flow cytometry, and immunohistochemistry. CRISPR-Cas9 was employed to delete CXCL12γ and the heparan sulfate (HS) co-polymerase EXT1 in BMSCs. To study the functional roles of BMSC-derived CXCL12γ and HSPGs in the interaction of MM cells with BMSCs cells, MM cell lines and primary MM cells were co-cultured with BMSCs. Results We observed that CXCL12γ is expressed in situ by reticular stromal cells in both normal and MM BM, as well as by primary BMSC isolates and BMSC lines. Importantly, upon secretion, CXCL12γ, unlike the CXCL12α isoform, was retained on the surface of BMSCs. This membrane retention of CXCL12γ is HSPG mediated, since it was completely annulated by CRISPR-Cas9-mediated deletion of the HS co-polymerase EXT1. CXCL12γ expressed by BMSCs and membrane-retained by HSPGs supported robust adhesion of MM cells to the BMSCs. Specific genetic deletion of either CXCL12γ or EXT1 significantly attenuated the ability of BMSCs to support MM cell adhesion and, in addition, impaired their capacity to protect MM cells from bortezomib-induced cell death. Conclusions We show that CXCL12γ is expressed by human BMSCs and upon secretion is retained on their cell surface by HSPGs. The membrane-bound CXCL12γ controls adhesion of MM cells to the stromal niche and mediates drug resistance. These findings designate CXCL12γ and associated HSPGs as partners in mediating MM–niche interaction and as potential therapeutic targets in MM.

scholarly journals The CXCL12gamma chemokine immobilized by heparan sulfate on stromal niche cells controls adhesion and mediates drug resistance in multiple myeloma

2020 ◽  
Author(s):  
Zemin Ren ◽  
Hildo Lantermans ◽  
Annemieke Kuil ◽  
Willem Kraan ◽  
Fernando Arenzana-Seisdedos ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Drug Resistance ◽  
Cell Adhesion ◽  
Heparan Sulfate ◽  
Stromal Cells ◽  
Unique Role ◽  
Bm Niche ◽  
Strong Adhesion ◽  
Membrane Bound ◽  
Cell Adhesion Molecule 1

AbstractThe homing/retention, survival and proliferation of multiple myeloma (MM) cells critically depends on interaction with CXCL12 expressing stromal cells in the bone marrow (BM) niche. Here, we report a unique role in this interaction for the recently characterized CXCL12gamma isoform, which contains an extended C-terminal domain that binds heparan-sulfate proteoglycans (HSPGs) with an extraordinary high affinity. We observed that CXCL12γ is expressed in situ by reticular stromal cells in both normal and MM BM, as well as by primary BM stromal-cell (BMSC) isolates and BMSC lines. Importantly, upon secretion, CXCL12γ, unlike the CXCL12α isoform, was retained on the surface of these BMSCs. This membrane retention of CXCL12γ is HSPG-mediated, since it was completely annulated by CRISPR-Cas9 mediated deletion of the heparan-sulfate (HS) co-polymerase EXT1. Recombinant CXCL12γ was found to induce strong adhesion of MM cells to vascular cell-adhesion molecule 1 (VCAM-1) coated plates. Furthermore, CXCL12γ expressed by BMSCs and membrane-retained by HSPGs, supported robust adhesion of MM cells to the BMSCs. Specific genetic deletion of either CXCL12γ or of EXT1 significantly attenuated the ability of BMSCs to support MM cell adhesion and, in addition, impaired their capacity to protect MM cells from bortezomib-induced cell death. Our data indicate that CXCL12γ functions as a membrane-bound ‘niche chemokine’, which plays a unique role in the interaction of MM cells with the stromal niche by controlling adhesion/retention as well as cell adhesion-mediated drug resistance (CAM-DR). These findings designate CXCL12γ and associated HSPGs as potential therapeutic targets in MM.

scholarly journals Reovirus-induced cell-mediated immunity for the treatment of multiple myeloma within the resistant bone marrow niche

2021 ◽  
Vol 9 (3) ◽  
pp. e001803
Author(s):  
Louise M E Müller ◽  
Gemma Migneco ◽  
Gina B Scott ◽  
Jenny Down ◽  
Sancha King ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
Immune Responses ◽  
Stromal Cells ◽  
Tumor Burden ◽  
Effector Memory ◽  
In Vivo Model ◽  
Bm Niche

BackgroundMultiple myeloma (MM) remains an incurable disease and oncolytic viruses offer a well-tolerated addition to the therapeutic arsenal. Oncolytic reovirus has progressed to phase I clinical trials and its direct lytic potential has been extensively studied. However, to date, the role for reovirus-induced immunotherapy against MM, and the impact of the bone marrow (BM) niche, have not been reported.MethodsThis study used human peripheral blood mononuclear cells from healthy donors and in vitro co-culture of MM cells and BM stromal cells to recapitulate the resistant BM niche. Additionally, the 5TGM1-Kalw/RijHSD immunocompetent in vivo model was used to examine reovirus efficacy and characterize reovirus-induced immune responses in the BM and spleen following intravenous administration. Collectively, these in vitro and in vivo models were used to characterize the development of innate and adaptive antimyeloma immunity following reovirus treatment.ResultsUsing the 5TGM1-Kalw/RijHSD immunocompetent in vivo model we have demonstrated that reovirus reduces both MM tumor burden and myeloma-induced bone disease. Furthermore, detailed immune characterization revealed that reovirus: (i) increased natural killer (NK) cell and CD8+ T cell numbers; (ii) activated NK cells and CD8+ T cells and (iii) upregulated effector-memory CD8+ T cells. Moreover, increased effector-memory CD8+ T cells correlated with decreased tumor burden. Next, we explored the potential for reovirus-induced immunotherapy using human co-culture models to mimic the myeloma-supportive BM niche. MM cells co-cultured with BM stromal cells displayed resistance to reovirus-induced oncolysis and bystander cytokine-killing but remained susceptible to killing by reovirus-activated NK cells and MM-specific cytotoxic T lymphocytes.ConclusionThese data highlight the importance of reovirus-induced immunotherapy for targeting MM cells within the BM niche and suggest that combination with agents which boost antitumor immune responses should be a priority.

scholarly journals ABCG2 expression, function, and promoter methylation in human multiple myeloma

Blood ◽  
2006 ◽  
Vol 108 (12) ◽  
pp. 3881-3889 ◽  
Author(s):  
Joel G. Turner ◽  
Jana L. Gump ◽  
Chunchun Zhang ◽  
James M. Cook ◽  
Douglas Marchion ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Drug Resistance ◽  
Protein Expression ◽  
Cell Lines ◽  
Promoter Methylation ◽  
Plasma Cells ◽  
Cancer Resistance ◽  
Response To Chemotherapy ◽  
Mrna And Protein Expression

AbstractWe investigated the role of the breast cancer resistance protein (BCRP/ABCG2) in drug resistance in multiple myeloma (MM). Human MM cell lines, and MM patient plasma cells isolated from bone marrow, were evaluated for ABCG2 mRNA expression by quantitative polymerase chain reaction (PCR) and ABCG2 protein, by Western blot analysis, immunofluorescence microscopy, and flow cytometry. ABCG2 function was determined by measuring topotecan and doxorubicin efflux using flow cytometry, in the presence and absence of the specific ABCG2 inhibitor, tryprostatin A. The methylation of the ABCG2 promoter was determined using bisulfite sequencing. We found that ABCG2 expression in myeloma cell lines increased after exposure to topotecan and doxorubicin, and was greater in logphase cells when compared with quiescent cells. Myeloma patients treated with topotecan had an increase in ABCG2 mRNA and protein expression after treatment with topotecan, and at relapse. Expression of ABCG2 is regulated, at least in part, by promoter methylation both in cell lines and in patient plasma cells. Demethylation of the promoter increased ABCG2 mRNA and protein expression. These findings suggest that ABCG2 is expressed and functional in human myeloma cells, regulated by promoter methylation, affected by cell density, up-regulated in response to chemotherapy, and may contribute to intrinsic drug resistance.

Hypoxia Induces Up-Regulation Of P-Glycoprotein and Promotes Resistance To Carfilzomib In Multiple Myeloma

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4907-4907
Author(s):  
Joseph Abraham ◽  
Salama N Noha ◽  
Abdel Kareem Azab
Keyword(s):  
Multiple Myeloma ◽  
Drug Resistance ◽  
Plasma Cells ◽  
Conflicts Of Interest ◽  
Tumor Hypoxia ◽  
Hematologic Malignancies ◽  
Multi Drug Resistance ◽  
Resistance Proteins ◽  
Hypoxic Conditions ◽  
Bm Niche

Abstract Introduction Multiple myeloma (MM) is a malignant neoplastic cancer of plasma cells that involves the bone marrow. Generally, patients will respond to treatment initially, but they later become resistant to therapy, and this is ultimately due to a change in the biology of the tumor. Multi-drug-resistance transporter proteins were shown to play a role in drug resistance in MM patients; P-glyco-protein (P-gp) is the most studied of the multi-drug resistance proteins, and it becomes up-regulated in response to many chemotheries. Hypoxia was shown to develop in the BM niche during progression of MM and to play a major role in the dissemination of MM cells to the new BM niches. Tumor-hypoxia was shown todevelop many kinds of solid tumors and hematologic malignancies. Specifically, hypoxia was shown to develop in the BM niche during progression of MM and to play a major role in the dissemination of MM cells to the new BM niches. In this study, we examinned the effect of hypoxia on the expression and activity of P-gp in MM and its contributing to drug resistance to therapies used in MM. Methods and Results We tested the effect of hypoxia on the activity of P-gp in MM lines. We incubated MM cells under hypoxic and normoxic conditions, and we tested their ability to pump out Rhodamine (Rh) by measuring Rh content in the cells by fluorescent reader. First, we optimized the concentration of Rh and the time of incubation with the cells. We found that at all concentrations tested (0.1, 0.5, 1, 5 and 10 ug/ml) and at all incubation time of cells with Rh with MM cells (0.25, 0.5, 1, 2, 4, 6, 8 and 24hrs) , hypoxia increased the efflux of Rh. The most significant efflux was achieved when incubating the cells for 1hr with Rh 1ug/ml. We found that hypoxia increased the efflux of Rh in all MM cell lines tested. Incubation of RPMI cells under hypoxic for 24hrs and 48hrs decreased the Rh content of the cells by about 40% and 65%, respectively. Carfilzomib was previously reported to be a substrate of P-gp, we tested the effect of carfilzomib on the efflux of Rd in the MM cells. Hypoxic and normoxic MM cells were treated for 5hrs with carfilzomib (5 nM) and then incubated for 1hrs with Rh (1ug/ml). We tested the Rh content of the cells by fluorescent reader and found that carfilzomib competed with Rh on the P-gp and decreased the efflux of Rh induced by hypoxic. We tested the effect of carfilzomib on induction of P-gp in hypoxic and normoxic MM cells by treating RPMI cells with a low dose of carfilzomib (0.25nM) for 48hrs under hypoxic or normoxic conditions, and tested the cells ability to efflux Rh. We found that carfilzomib increased P-gp expression and induced efflux of about 30% of the Rh in non-treated normoxic cells.  Hypoxia induced efflux of about 65% of normoxic cells, but no effect was observed with the treatment of carfilzomib. Furthermore, we tested the hypoxia-induced P-gp expression in MM on the sensitivity of MM cells to carfilzomib. We incubated MM cells for 24hrs in hypoxic and normoxic conditions, and cells were treated with carfilzomib (0 or 5nM) for additional 24hrs. We found that while carfilzomib induced the death of about 40% of the cells under normoxic condition, it had no significant effect on the survival of MM cell under hypoxic conditions. Conclusion Hypoxia induced a significant up-regulation of P-gp in MM cells, and increased MM drug resistance to carfilzomib. These results provide mechanistic evidence for drug resistance to carfilzomib in MM, and suggest hypoxia as a novel therapeutic to prevent upregulation of P-gp and drug resistance. Disclosures: No relevant conflicts of interest to declare.

Bone Marrow Stromal Cells Induce Bortezomib Resistance in Multiple Myeloma Cells through Downregulation of miRNA-101-3p Targeting Survivin

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1772-1772 ◽  
Author(s):  
Jahangir Abdi ◽  
Yijun Yang ◽  
Patrick Meyer-Erlach ◽  
Hong Chang
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Drug Resistance ◽  
Cell Lines ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Molecular Mechanisms ◽  
Marrow Stromal Cells ◽  
Survivin Gene ◽  
Induced Apoptosis

Abstract INTRODUCTION It is not yet fully understood how bone marrow microenvironment components especially bone marrow stromal cells (BMSCs) induce drug resistance in multiple myeloma (MM). This form of drug resistance has been suggested to pave the way for intrinsic (de novo) resistance to therapy in early stages of the disease and contribute to acquired drug resistance in the course of treatment. Hence, deciphering the molecular mechanisms involved in induction of above resistance will help identify potential therapeutic targets in MM combined treatments. Our previous work showed that BMSCs (normal and MM patient-derived) induced resistance to bortezomib (BTZ) compared with MM cells in the absence of stroma. This resistance was associated with modulation of a transcriptome in MM cells, including prominent upregulation of oncogenes c-FOS, BIRC5 (survivin) and CCND1. However; whether these oncogenes mediate BTZ resistance in the context of BMSCs through interaction with miRNAs is not known. METHODS Human myeloma cell lines, 8226, U266 and MM.1s, were co-cultured with MM patient-derived BMSCs or an immortalized normal human line (HS-5) in the presence of 5nM BTZ for 24 h. MM cell monocultures treated with 5nM BTZ were used as controls. Co-cultures were then applied to magnetic cell separation (EasySep, Stem Cell Technologies) to isolate MM cells for downstream analyses (western blotting and qPCR). Total RNA including miRNAs was isolated from MM cell pellets (QIAGEN miRNeasy kit), cDNAs were synthesized (QIAGEN miScript RT II kit) and applied to miScript miRNA PCR Array (SABioscience, MIHS-114ZA). After normalization of all extracted Ct values to 5 different housekeeping genes, fold changes in miRNA expression were analyzed in co-cultures compared to MM cell monocultures using the 2-ΔΔCt algorithm. Moreover, survivin gene was silenced in MM cells using Ambion® Silencer® Select siRNA and Lipofectamine RNAiMAX transfection reagent. Survivin-silenced cells were then seeded on BMSCs and exposed to BTZ. Percent apoptosis of gated CD138+ MM cells was determined using FACS. For our overexpression and 3'UTR reporter experiments, we transiently transfected MM cells with pre-miR-101-3p, scrambled miRNA or pEZX-3'UTR constructs using Endofectin reagent (all from GeneCopoeia). RESULTS BMSCs upregulated survivin gene / protein (a member of inhibitors of apoptosis family) and modulated an array of miRNAs in MM cells compared to MM cells in the absence of stroma. The more noticeably downregulated miRNAs were hsa-miR-101-3p, hsa-miR-29b-3p, hsa-miR-32-5p, hsa-miR-16-5p (4-30 fold) and highly upregulated ones included hsa-miR-221-3p, hsa-miR-409-3p, hsa-miR-193a-5p, hsa-miR-125a-5p (80-330 fold). We focused on miRNA-101-3p as it showed the highest level of downregulation (30 fold) and has been shown to function as an important tumor suppressor in other malignancies. Real time RT-PCR confirmed downregulation of miRNA-101-3p. Moreover, microRNA Data Integration Portal (mirDIP) identified miRNA-101-3p as a putative target for survivin and Luciferase activity assays confirmed binding of miRNA-101-3p to 3'UTR of survivin. In addition, overexpression of miRNA-101-3p downregulated survivin and sensitized MM cells to BTZ-induced apoptosis. Furthermore, silencing of survivin upregulated miRNA-101-3p and increased BTZ-induced apoptosis in MM cell lines both in the absence of BMSCs (Apoptosis range in BTZ-treated conditions: 57.65% ± 4.91 and 28.66% ± 0.78 for si-survivin and scrambled control, respectively, p<0.05) and in the presence of BMSCs (41.23% ± 1.43 and 14.8% ± 0.66, for si-survivin and scrambled control, respectively, p<0.05). CONCLUSION Our results indicate that BMSCs downregulated miRNA-101-3p and upregulated survivin in MM cells compared to MM cells in the absence of stroma. Silencing of survivin or overexpression of miRNA-101-3p sensitized MM cells to BTZ in the presence of BMSCs. These findings suggest that miRNA-101-3p mediates BTZ response of MM cells in the presence of BMSCs by targeting survivin and disclose a role of survivin-miRNA-101-3p axis in regulation of BMSCs-induced BTZ resistance in MM cells, thus provide a rationale to further investigate the anti-myeloma activity of miRNA-101-3p in combination with BTZ as a potential novel therapeutic strategy in MM. Disclosures No relevant conflicts of interest to declare.

scholarly journals Bone Marrow Microenvironment in Multiple Myeloma Progression

2012 ◽  
Vol 2012 ◽  
pp. 1-5 ◽  
Author(s):  
S. Manier ◽  
A. Sacco ◽  
X. Leleu ◽  
I. M. Ghobrial ◽  
A. M. Roccaro
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Stromal Cells ◽  
Plasma Cells ◽  
Cellular Compartment ◽  
Cell Homing ◽  
Premetastatic Niche ◽  
Bm Niche ◽  
Malignant Plasma Cell ◽  
Made In

Substantial advances have been made in understanding the biology of multiple myeloma (MM) through the study of the bone marrow (BM) microenvironment. Indeed, the BM niche appears to play an important role in differentiation, migration, proliferation, survival, and drug resistance of the malignant plasma cells. The BM niche is composed of a cellular compartment (stromal cells, osteoblasts, osteoclasts, endothelial cells, and immune cells) and a noncellular compartment including the extracellular matrix (ECM) and the liquid milieu (cytokines, growth factors, and chemokines). In this paper we discuss how the interaction between the malignant plasma cell and the BM microenvironment allowed myeloma progression through cell homing and the new concept of premetastatic niche.

scholarly journals P-selectin glycoprotein ligand regulates the interaction of multiple myeloma cells with the bone marrow microenvironment

Blood ◽  
2012 ◽  
Vol 119 (6) ◽  
pp. 1468-1478 ◽  
Author(s):  
Abdel Kareem Azab ◽  
Phong Quang ◽  
Feda Azab ◽  
Costas Pitsillides ◽  
Brian Thompson ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Drug Resistance ◽  
Stromal Cells ◽  
Critical Role ◽  
Loss Of Function ◽  
Downstream Signaling ◽  
Target Organs ◽  
Regulation Of Growth

Abstract Interactions between multiple myeloma (MM) cells and the BM microenvironment play a critical role in the pathogenesis of MM and in the development of drug resistance by MM cells. Selectins are involved in extravasation and homing of leukocytes to target organs. In the present study, we focused on adhesion dynamics that involve P-selectin glycoprotein ligand-1 (PSGL-1) on MM cells and its interaction with selectins in the BM microenvironment. We show that PSGL-1 is highly expressed on MM cells and regulates the adhesion and homing of MM cells to cells in the BM microenvironment in vitro and in vivo. This interaction involves both endothelial cells and BM stromal cells. Using loss-of-function studies and the small-molecule pan-selectin inhibitor GMI-1070, we show that PSGL-1 regulates the activation of integrins and downstream signaling. We also document that this interaction regulates MM-cell proliferation in coculture with BM microenvironmental cells and the development of drug resistance. Furthermore, inhibiting this interaction with GMI-1070 enhances the sensitization of MM cells to bortezomib in vitro and in vivo. These data highlight the critical contribution of PSGL-1 to the regulation of growth, dissemination, and drug resistance in MM in the context of the BM microenvironment.

2-Methoxyestradiol overcomes drug resistance in multiple myeloma cells

Blood ◽  
2002 ◽  
Vol 100 (6) ◽  
pp. 2187-2194 ◽  
Author(s):  
Dharminder Chauhan ◽  
Laurence Catley ◽  
Teru Hideshima ◽  
Guilan Li ◽  
Richard Leblanc ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Drug Resistance ◽  
Stromal Cells ◽  
Growth Arrest ◽  
Peripheral Blood Lymphocytes ◽  
Leukemic Cells ◽  
Vascular Endothelial ◽  
Normal Peripheral Blood ◽  
Induced Apoptosis ◽  
Endothelial Growth Factor

Abstract 2-Methoxyestradiol (2ME2) an estrogen derivative, induces growth arrest and apoptosis in leukemic cells and is also antiangiogenic. In this study, we demonstrate that 2ME2 inhibits growth and induces apoptosis in multiple myeloma (MM) cell lines and patient cells. Significantly, 2ME2 also inhibits growth and induces apoptosis in MM cells resistant to conventional therapies including melphalan (LR-5), doxorubicin (Dox-40 and Dox-6), and dexamethasone (MM.1R). In contrast to its effects on MM cells, 2ME2 does not reduce the survival of normal peripheral blood lymphocytes. Moreover, 2ME2 enhances Dex-induced apoptosis, and its effect is not blocked by interleukin-6 (IL-6). We next examined the effect of 2ME2 on MM cells in the bone marrow (BM) milieu. 2ME2 decreases survival of BM stromal cells (BMSCs), as well as secretion of vascular endothelial growth factor (VEGF), and IL-6 triggered by the adhesion of MM cells to BMSCs. We show that apoptosis induced by 2ME2 is mediated by the release of mitochondrial cytochrome-c (cyto-c) and Smac, followed by the activation of caspases-8, -9, and -3. Finally, 2ME2 inhibits MM cell growth, prolongs survival, and decreases angiogenesis in a murine model. These studies, therefore, demonstrate that 2ME2 mediates anti-MM activity directly on MM cells and in the BM microenvironment. They provide a framework for the use of 2ME2, either alone or in combination with Dex, to overcome drug resistance and to improve outcome in MM.

scholarly journals CHEK1 and circCHEK1_246aa Promote Multiple Myeloma Malignancy By Evoking Chromosomal Instability and Bone Lesion

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 9-10
Author(s):  
Ye Yang ◽  
Chunyan Gu ◽  
Wang Wang ◽  
Xiaozhu Tang
Keyword(s):  
Multiple Myeloma ◽  
Cell Proliferation ◽  
Drug Resistance ◽  
Chromosomal Instability ◽  
Cellular Proliferation ◽  
Plasma Cells ◽  
Osteoclast Differentiation ◽  
Bone Lesion ◽  
Catalytic Center ◽  
Bm Niche

Key findings CHEK1 and circCHEK1_246aa induce multiple myeloma cell proliferation, drug resistance, and bone lesion formation CHEK1 and circCHEK1_246aa evoke myeloma chromosomal instability, partially through CEP170 activation Abstract Multiple myeloma (MM) is characterized by clonal expansion of plasma cells in the bone marrow (BM). Therefore, effective therapeutic interventions must target both myeloma cells and the BM niche. In the present study, we first demonstrated that CHEK1 expression was significantly increased in human MM samples relative to normal plasma cells, and that in MM patients, high CHEK1 expression was associated with poor outcomes. CHEK1 overexpression increased cellular proliferation in MM cells and evoked drug resistance in vitro, while CHEK1 knockdown abrogated this effect. Moreover, CHEK1 was a high-risk gene for poor outcome in MM patients, and, in paired samples from MM patients taken from newly diagnosed and relapsed MM, CHEK1 expression was upregulated. CHEK1-mediated increases in cell proliferation and drug resistance were due in part to CHEK1-induced chromosomal instability (CIN), as demonstrated by Giemsa staining, exon sequencing, and immunofluorescence. CHEK1 activated CIN, partly by phosphorylating CEP170. Interestingly, CHEK1 promoted osteoclast differentiation by direct phosphorylation and activation of NFATc1, indicating that CHEK1 inhibition could target both MM cell proliferation and macrophage osteoclast differentiation in the BM niche. Intriguingly, we also discovered that MM cells expressed circCHEK1_246aa, a circular CHEK1 RNA, which encoded and was translated to the CHEK1 kinase catalytic center. Transfection of circCHEK1_246aa increased MM CIN and osteoclast differentiation similarly to CHEK1 overexpression, suggesting that MM cells could secrete circCHEK1_246aa in the BM niche to increase the invasive potential of MM cells and promote osteoclast differentiation. Finally, we demonstrated in vivo in xenograft models that CHEK1 overexpression prompted MM proliferation and drug resistance, while CHEK1 knockdown conversely inhibited MM growth. Together, these findings suggest that targeting the enzymatic catalytic center encoded by CHEK1 mRNA and circCHEK1_246aa is a promising therapeutic modality to target both MM cells and the BM niche. Figure Disclosures No relevant conflicts of interest to declare.

Higher Expression Levels of the Adhesion Molecules VLA-4 and ICAM-1 on Multiple Myeloma Cells after Chemotherapy Are Associated with Survival of a Multidrug Resistant Subpopulation in Vivo.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4866-4866
Author(s):  
Ralf Schmidmaier ◽  
Kerstin Mörsdorf ◽  
Philipp Baumann ◽  
Bertold Emmerich ◽  
Gerold Meinhardt
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Drug Resistance ◽  
Adhesion Molecules ◽  
Stromal Cells ◽  
High Dose ◽  
Myeloma Cells ◽  
Expression Levels ◽  
Before And After ◽  
Primary Drug

Abstract Objectives: Primary drug resistance is a major problem in multiple myeloma (MM), an incurable disease of the bone marrow. Adhesion of multiple myeloma cells to bone marrow stromal cells (BMSC) has been shown to cause strong primary resistance. The adhesion molecules LFA-1 and VLA-4 are upregulated upon treatment with cytotoxic agents. Furthermore, we have shown that the corresponding ligands on HS-5 BMSCs, VCAM-1 and ICAM-1, are upregulated after incubation with melphalan, suggesting increase of adhesion mediated drug resistance after chemotherapy. In this context, the expression levels of important adhesion molecules on MM cells of consecutive MM patients before and after chemotherapy have been determined in this study. Methods: The expression levels of VLA-1, VLA-4, VLA-5, LFA-1, VCAM, ICAM-1, CD138, CD38, and CD56 were determined on MM cell lines, HS-5 stromal cells, and primary myeloma cells of 20 consecutive patients by flow cytometry in comparison to isotype control. 9 patients had been pre-treated (mostly induction chemotherapy and high dose melphalan with stem cell rescue) and 11 patients had been at diagnosis without treatment. Interpatient comparison of treated and untreated patients was performed. Intrapatient analysis (before and after high dose chemotherapy) will be performed in the follow up. Results: VLA-4 and ICAM-1 are upregulated after chemotherapy by 54% and 64%, respectively. Similar upregulation of CD38 could be observed (62%), whereas CD138 shows downregulation by about 50%. CD56, VCAM, and LFA-1 expression was not significantly altered after chemotherapy. Conclusion: The adhesion molecules VLA-4 and ICAM-1, which are essential for MM-BMSC interaction, are upregulated after chemotherapy. This finding supports our preclinical data and the hypothesis, that adhered, primary drug resistant MM cells are selected by chemotherapy and herewith contribute to multidrug resistance in multiple myeloma.

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