scholarly journals APOBEC3B Induction Following DNA Damage Response Modulates the Survival and Treatment Response in Human Multiple Myeloma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4381-4381
Author(s):  
Lijie Xing ◽  
Jiye Liu ◽  
Yuyin Li ◽  
Liang Lin ◽  
Kenneth Wen ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Dna Damage ◽  
Protein Expression ◽  
Treatment Response ◽  
Cell Lines ◽  
Critical Role ◽  
Dependent Manner ◽  
Dna Damages ◽  
Drug Responses

Apolipoprotein B mRNA editing catalytic polypeptide-like 3B (APOBEC3B, A3B) is one of 7-membered DNA cytosine deaminase family, causing cytosine-to-uracil (C-to-U) deamination in single-stranded DNA and promoting mutations in multiple human cancers including multiple myeloma (MM). High APOBEC3B expression is found in a significant portion of MM patients with MAF overexpression among t(14;16) and t(14;20). A3B upregulation is further associated with poor prognosis in MM, suggesting its role in the MM pathophysiology. However, approximately 23% MM patients with high APOBEC3 activity are associated with MAF/MAFA/MAFB translocations, the remainder of patients with high APOBEC3 carry neither translocations nor overexpression of these genes. Besides, studies are lacking on how A3B is regulated and the role of A3B in drug responses in MM. We here defined new mechanisms controlling A3B expression and further characterized its impact on treatment responses to current anti-MM therapies. Using qRT-PCR, A3B transcript is significantly higher than other members of the APOBEC3 gene family in MM cell lines (n=19) and MM patients, indicating that A3B may play a major role in MM. Using immunoblotting analysis, A3B protein expression was further confirmed in MM cell lines with various levels (n=10). Importantly, A3B mRNA upregulation by 1.34-42.64 folds was observed in CD138-purified cells from majorities of MM patients (83.3%) when compared to PBMC from the same individual (n=12). In MM cell lines without MAF/MAFA/MAFB translocation as a study model, higher A3B protein expression is associated with higher DNA damage levels as evidenced by higher γ-H2AX. These results suggest that A3B expression might be influenced by DNA damage levels in MM cells. Following a short time treatment of gamma-irradiation to cause DNA damages, A3B expression in viable MM cells was enhanced in a dose-dependent manner. We next treated MM cells (n=5) with common anti-MM drugs such as Melphalan (Mel) and Bortizomib (btz), both of which induce DNA damages, followed by examination of changes in A3B and γ-H2AX. Under sublethal treatment conditions of Mel or btz, A3B was consistently induced at both mRNA and protein levels in multiple MM cell lines regardless of the baseline A3B expression. Significantly, A3B was upregulated and associated with increased γ-H2AX in patient MM cells treated with Mel or btz under sub-lethal doses. Since DNA damages activate the ATR/ATM pathway, we next investigated whether these kinases mediate A3B induction following treatments with these compounds in MM cells. The presence of ATM or ATR inhibitors blocked A3B upregulated by these DNA damage-inducing treatments in MM cell lines (n=3), indicating an ATM/ATR-dependent pathway for A3B changes. Next, gene-specific CRISPR knock out (KO) and inducible-shRNA knockdown (KD) were used to determine the functional impact of perturbation of A3B in proliferation and survival of MM cells. Both KO and KD of A3B decreased growth and viability of MM cell lines regardless of sensitive or resistant to dexamethasone or lenalidomide. Using LIVE/DEAD fixable Aqua Stain and annexin V-based flow cytometric analysis, A3B inhibition enhanced growth arrest followed by apoptosis in MM cells. Significantly, A3B KD by its shRNA in RPMI8226 MM cells enhanced sensitivity to pomalidomide. Taken together, these data indicate that increased A3B level plays a critical role in MM cell survival and drug responses. DNA damages triggered by IR, Mel, or btz further enhance A3B expression via ATM/ATR pathway, which in turn increases subclonal diversity leading to drug resistance. The role of A3B in disease pathophysiology and progression, coupled with its function in mediating treatment response, suggest potential utility of targeting A3B in MM. Disclosures Munshi: Celgene: Consultancy; Abbvie: Consultancy; Oncopep: Consultancy; Adaptive: Consultancy; Amgen: Consultancy; Janssen: Consultancy; Takeda: Consultancy. Anderson:Sanofi-Aventis: Other: Advisory Board; Bristol-Myers Squibb: Other: Scientific Founder; Oncopep: Other: Scientific Founder; Amgen: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau.

Targeting Deubiquitylating Enzyme USP1 in Multiple Myeloma

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1813-1813
Author(s):  
Deepika Sharma Das ◽  
Yan Song ◽  
Arghya Ray ◽  
Paul Richardson ◽  
Dharminder Chauhan ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Dna Repair ◽  
Cell Lines ◽  
Cancer Cell ◽  
Therapeutic Index ◽  
Cysteine Proteases ◽  
Dependent Manner ◽  
Healthy Donors ◽  
Reporter Assays

Abstract Introduction Proteasome inhibitor Bortezomib is effective therapy of relapsed/refractory and newly diagnosed multiple myeloma (MM); however, dose-limiting toxicities and the development of resistance limit its long-term utility. Importantly, the ability of bortezomib to overcome resistance to conventional therapies has validated therapeutically targeting the Ubiquitin Proteasome System (UPS), and suggested potential utility of inhibitors of other components of the UPS including deubiquitylating enzymes (DUBs). Therapeutic strategies directed against DUBs may allow for more specific targeting of the UPS, and therefore be less likely to have off-target activities with associated toxicities. Our prior studies have identified a role of USP7, USP14, and UCHL5 in MM pathogenesis, and provided the rationale for targeting these DUBs in MM (Chauhan et al., Cancer Cell 2012, 11:345-358; Tian et al., Blood 2014, 123:706-716). Among DUBs, USP1 regulates DNA repair and the Fanconi anemia pathway through its association with its WD40 binding partner UAF1, and through its deubiquitylation of two critical DNA repair proteins, FANCD2-Ub and PCNA-Ub. Here we examined the role of USP1 DUB in MM using both biochemical and RNA interference strategies. Methods We utilized MM cell lines, patient cells, and peripheral blood mononuclear cells (PBMCs) from normal healthy donors. Cell viability was assessed using WST and CellTiter-Glo assays. MM.1S MM cells were transiently transfected with control short interfering RNA (siRNA), USP1 ON TARGET plus SMART pool siRNA using the cell line Nucleofector Kit V. A biochemical inhibitor of USP1 SJB3-019A (SJB) was purchased from Medchem Express, USA. In vitro DUB enzymatic activity was assessed using Ubiquitin-AMC and Ubiquitin-Rhodamine assay kits, as well as Ub-CHOP-reporter and K48-linked Ubiquitin tetramers. Competitive Ub-VS probe labeling was performed, as previously described (Chauhan et al., Cancer Cell 2012, 11:345-358). Signal transduction pathways were evaluated using immunoblotting. Statistical significance of data was determined using a Student's t test. Results Immunoblot analyses show higher USP1 levels in MM cell lines and patient cells than normal cells.USP1-siRNA inhibited MM cell proliferation, which was rescued by transfection of USP1 (WT). Using Ub-Rhodamine, Ub-AMC, and Ub-EKL reporter assays, we found higher USP1 deubiquitylating activity in patient MM cells versus normal cells, suggesting a favorable therapeutic index for targeting USP1. Importantly, siRNA-knockdown of USP1 both promoted degradation of tumorigenic ID1 protein, and inhibited proliferation of bortezomib-resistant (ANBL-6.BR) MM cells, suggesting that novel agents targeting USP1 may overcome bortezomib resistance. We next examined the effects of USP1 inhibitor SJB3 on MM cell growth and survival in our models of MM. Analysis using Ub-Rhodamine, Ub-AMC, and Ub-EKL reporter assays in a panel of MM cell lines showed that SJB is a potent, specific, and selective inhibitor of USP1 DUB activity (EC50 = 50 ± 5.7 nM), which does not inhibit other DUBs (USP2/USP5/USP7/USP14) or other families of cysteine proteases (EC50>100 μM). SJB blocks labeling of USP1 with HA-Ub-VS probe in a concentration-dependent manner, but did not alter labeling of other DUBs with HA-Ub-VS. SJB inhibits USP1-mediated cleavage of K48 linked polyubiquitin chains, but not that mediated by USP2 or USP7. Treatment of MM cell lines (MM.1S, MM.1R, RPMI-8226, Dox-40, ARP1, KMS11, U266, ANBL6.WT, ANBL6.BR, and LR5) and primary patient cells for 24h significantly decreases their viability (IC50 range 100nM to 500nM) (p < 0.05; n=3) without markedly affecting PBMCs from normal healthy donors, suggesting specific anti-MM activity and a favorable therapeutic index for SJB. Tumor cells from 3 of 5 patients were obtained from patients whose disease was progressing while on bortezomib, dexamethasone, and lenalidomide therapies. Mechanistic studies show that SJB-triggered apoptosis is associated with degradation of USP1 and Id1 protein. Finally, combination of SJB with lenalidomide, pomalidomide, HDACi ACY-1215, or bortezomib both induces synergistic anti-MM activity and overcomes drug resistance. Conclusion Our preclinical studies provide the framework for clinical evaluation of USP1 inhibitors, alone or in combination, as a potential MM therapy. Disclosures Chauhan: Stemline Therapeutics: Consultancy.

Role Of Sirtuin-6 In Maintenance Of Genomic Instability In Multiple Myeloma Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 276-276
Author(s):  
Michele Cea ◽  
Antonia Cagnetta ◽  
Mariateresa Fulciniti ◽  
Yu-Tzu Tai ◽  
Chirag Acharya ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Dna Damage ◽  
Dna Repair ◽  
Genomic Instability ◽  
Cell Lines ◽  
Dsb Repair ◽  
Dna Damaging Agents ◽  
Genotoxic Agents ◽  
Equity Ownership

Abstract Background Deregulation of the DNA damage response (DDR) signaling machinery underlies genomic instability, leading to cancer development and clonal evolution. Multiple Myeloma (MM) remains an incurable disease characterized by a highly unstable genome, with aneuploidy observed in nearly all patients. The mechanism causing this karyotypic instability is largely unknown, but recent observations have correlated these abnormalities with dysfunctional DDR machinery. Mammalian NAD+-dependent deacetylase sirtuin-6 (SIRT6) is emerging as new protein involved in multiple pathways, including maintenance of genome integrity. Methods A panel of 18 MM cell lines, both sensitive and resistant to conventional and novel anti-MM therapies, was used in this study. Blood and BM samples from healthy volunteers and MM patients were obtained after informed consent and mononuclear cells (MNCs) separated by Ficoll-Paque density sedimentation. Patient MM cells were isolated from BM MNCs by CD138-positive selection. Lentiviral delivery was used for expression and knock-down of SIRT6 in MM cell lines. The biologic impact of SIRT6 phenotype was evaluated using cell growth, viability and apoptosis assays. DNA Double-Strand Breaks (DSB) repair occurring via homologous recombination (HR) or non-homologous end-joining (NHEJ) pathways was assessed using a transient direct repeat (DR)-GFP/I-SceI system. Results A comparative gene expression analysis of 414 newly-diagnosed uniformly-treated MM patients showed high levels of SIRT6 mRNA in MM patients versus MGUS or normal donors; moreover, in active MM elevated SIRT6 expression correlated with adverse clinical outcome. Due to its prognostic significance, we further evaluated its role in MM biology. We found higher SIRT6 nuclear expression in MM cell lines and primary cells compared to PBMCs from healthy donors. Targeting SIRT6 by specific shRNA increased MM cell survival by reducing DNA repair efficiency (HR and NHEJ). Whole genome profiling of three different SIRT6 knockout (Sirt6-/-) MM cell lines identified a restricted effect of SIRT6 silencing on transcription of DNA damage genes, which also represented the most down-regulated genes. Consistent with these data, GSEA algorithm revealed that gene set regulating DNA repair were prominently enriched in SIRT6 depleted cells (p<0.0001 and FDR=0.003), confirming the role of SIRT6 in this pathway. We next examined the therapeutic relevance of SIRT6 inhibition in MM by evaluating the effect of SIRT6 depletion on cytotoxicity induced by genotoxic agents. SIRT6 shRNA impaired DNA DSB repair pathways triggered by DNA damaging agents, thereby enhancing overall anti-MM activity of these agents. Finally, in concert with our in vitro data, studies using our human MM xenograft model confirmed that SIRT6 depletion enhanced anti-MM activity of DNA-damaging agents. Conclusion Collectively, our data provide basis for targeting SIRT6 as a novel therapeutic strategy in combination with genotoxic agents to enhance cytotoxicity and improve patient outcome in MM. Disclosures: Tai: Onyx: Consultancy. Hideshima:Acetylon Pharmaceuticals: Consultancy. Chauhan:Vivolux: Consultancy. Anderson:celgene: Consultancy; onyx: Consultancy; gilead: Consultancy; sanofi aventis: Consultancy; oncopep: Equity Ownership; acetylon: Equity Ownership.

The Biologic Relevance of PIM Kinases in the Context of Multiple Myeloma and Their Potential As Therapeutic Targets in Combination Drug Therapy

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2992-2992
Author(s):  
Janani Ramachandran ◽  
Loredana Santo ◽  
Homare Eda ◽  
Dharminder Chauhan ◽  
Ka Tat Siu ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Dna Damage ◽  
Drug Therapy ◽  
Cell Viability ◽  
Cell Lines ◽  
Research Funding ◽  
Doxorubicin Treatment ◽  
Pim Kinases

Abstract The proto-oncogene proviral integration sites for moloney murine leukemia virus (PIM) are serine/threonine kinases currently under investigation as therapeutic targets in hematologic cancers, where they have important functional roles as mediators of apoptosis, cell migration and homing. Thus far in Multiple Myeloma (MM), the PIM Kinases have been shown to mediate cap-dependent and cap-independent translation through the PI3K/AKT pathway and the activation of mTOR signaling (Lu J et al. 2013). In the context of the bone marrow microenvironment, PIM kinase expression in MM cells is known to be elevated in the presence of bone marrow stromal cells, and is mediated by the IL-6/STAT3 pathway, as well as the TNFα/NFΚB pathway (activated by Osteoclast secretion) (Hiasa M et al. 2014). Here, we have further studied the role of PIM kinases in MM. We observed elevated expression of PIM 1, 2 and 3 in patient derived myeloma cells (CD138+) as opposed to the stromal compartment (CD138-), confirming that PIM expression is predominantly hematologic lineage-specific. Inhibition of all three kinases by a pan PIM inhibitor results in reduced cell viability in tested human-derived MM cell lines, reinforcing the importance of the kinases as targets in drug therapy, as shown previously. Because the expression of Pim2 is consistently higher in primary tumor cells as well as in human MM lines, compared to Pim1 and Pim3, we sought to identify their distinct biologic significance. Single knock down (KD) of each kinase resulted in varying effects on cell viability, suggesting, together with the PIM expression profile, that the three kinases play different roles in the biology of MM. As the Pim2 KD resulted in the most profound decrease in cell viability, we focused our efforts to dissect the mechanistic importance of Pim2. Since Pim1 and Pim2 regulate the DNA damage response (DDR) via checkpoint kinase 1 (Chk1) in other hematologic malignancies, we investigated the effect of Pim2 KD on major signaling factors involved in the DDR. Transient KD resulted in phosphorylation of DDR pathway markers including ATR, CHK1/2, P21 and H2AX, and mimicked the effects of Doxorubicin treatment (a known DNA Damage causing agent). Furthermore, Doxorubicin treatment downregulated Pim2 expression, suggesting that Pim2 functions as an upstream regulator of the DDR pathway in MM. Pim2 appears to be the most relevant target in MM; however, because of a lack of a specific PIM2 inhibitor we used the pan-PIM inhibitor as a tool compound. Although the pan PIM kinase inhibitor showed single agent activity, combination approaches were more efficacious. Combining the pan-PIM inhibitor with bortezomib shows a significant synergistic effect on cell viability in multiple MM cell lines (MM1S, U266, KMS-12BM). Based on our KD experiments Pim2 predominantly mediates cell viability, suggesting that specifically targeting Pim2 in combination with Bortezomib will have a more direct effect on MM cell survival. To confirm the role of Pim2, ongoing experiments aim to focus on the effects of ectopically expressing Pim2 in the context of myeloma. We propose that Pim2 overexpression will not only serve anti-apoptotic purposes, but will further protect against DNA Damage in human MM cell lines. Disclosures Chauhan: Stemline Therapeutics: Consultancy. Huszar:Astra Zeneca: Employment. Raje:AstraZeneca: Research Funding; Onyx: Consultancy; BMS: Consultancy; Acetylon: Research Funding; Millenium: Consultancy; Amgen: Consultancy; Novartis: Consultancy; Takeda: Consultancy; Eli Lilly: Research Funding; Celgene Corporation: Consultancy.

The xCT Antiporter Is a Therapeutic Target in the ABC Subtype of DLBCL

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3996-3996
Author(s):  
Xiaolei Wei ◽  
Yun Mai ◽  
Ru Feng ◽  
B. Hilda Ye
Keyword(s):  
Oxidative Stress ◽  
Cell Death ◽  
B Cell ◽  
Cell Lines ◽  
Critical Role ◽  
B Cell Lymphoma ◽  
Dependent Manner ◽  
Ros Accumulation

Abstract Diffuse large B cell lymphoma (DLBCL) is the most common lymphoid malignancy in the adult population and can be subdivided into two main subtypes, i.e. GCB-DLBCL and ABC-DLBCL. While both subtypes are derived from normal germinal center (GC) B cells, they differ in B cell maturation stage, transformation pathway, and clinical behavior. When treated with either the combination chemotherapy CHOP or the immuno-chemotherapy R-CHOP, the survival outcome of ABC-DLBCL patients is typically much worse than that of GCB-DLBCL patients. Although the molecular mechanisms underlying this survival disparity remain poorly understood, an attractive hypothesis is that there exist subtype-specific resistance mechanisms directed against the chemo-therapy drugs in the original CHOP formulation. In support of this notion, our previous study has revealed that Doxorubicin (Dox), the main cytotoxic ingredient in CHOP, has subtype-specific mechanisms of cytotoxicity in DLBCLs due to differences in its subcellular distribution pattern. In particular, Dox-induced cytotoxicity in ABC-DLBCLs is largely dependent on oxidative stress rather than DNA damage response. Based on these findings, we hypothesize that agents capable of disturbing the redox balance in ABC-DLBCL cells could potentiate the therapeutic activity of first line lymphoma therapy. As the major route of cystine uptake from extracellular space, the xCT cystine/glutamate antiporter controls the rate-limiting step for glutathione (GSH) synthesis in several types of cancer cells, including CLL. We focused the current study on xCT because its protein stability is known to be positively regulated by a splicing variant of CD44 and we have recently published that expression of CD44 and CD44V6 are poor prognosticators for DLBCL. Indeed, we found that surface CD44 is exclusively expressed in ABC-DLBCL (6/6) but not GCB-DLBCL (0/5) cell lines. In addition, the xCT proteins in two ABC-DLBCL cell lines, Riva and SuDHL2, are extraordinarily stable, with half-lives exceeding 24 hours. As such, transient transfection using siRNA oligos was ineffective in reducing the endogenous xCT protein in ABC-DLBCL cell lines. To circumvent this issue, we turned to a clinically approved anti-inflammatory drug, sulfasalazine (SASP), which is a validated xCT inhibitor in its intact form. When Riva and SuDHL2 cells were treated overnight with the IC50 dose of SASP, the endogenous GSH pool was drastically reduced, leading to significant increase in intracellular ROS, p38 and JNK activation, and progressive apoptosis. Unexpectedly, we found that Dox-treated cells had significantly elevated GSH levels, possibly the result of an antioxidant response to Dox-triggered ROS accumulation. This increase in GSH was completely suppressed when the IC25 dosage of SASP was included in the Dox treatment. As expected, SASP/Dox combination significantly enhanced Dox-triggered ROS accumulation and synergistically promoted cell death in Riva and SuDHL2 cells. Mechanistically, p38 activation and cell death induced by SASP/Dox combination could be markedly attenuated by pretreatment with glutathione monoethyl ester, demonstrating the critical role of oxidative stress. Furthermore, cytotoxicity triggered by SASP/Dox could also be suppressed by the p38 inhibitor, SB203580. We have developed stable cell lines expressing xCT shRNA to confirm the results obtained with SASP. In vivo interactions between SASP and Dox are also being evaluated in xenograft-based ABC-DLBCL models. In summary, we report here for the first time a critical role of xCT in sustaining in vivo GSH production in ABC-DLBCL cells. More importantly, pharmacologic inhibition of xCT function in ABC-DLBCL cells not only prevented Dox-induced endogenous GSH increase, but also potentiated Dox-induced ROS accumulation and cytotoxicity in a p38-dependent manner. With additional evidence from ongoing experiments, our study aims to provide a mechanistic basis for development of novel therapies that target either xCT or redox homeostasis to improve treatment outcomes for ABC-DLBCLs. Disclosures No relevant conflicts of interest to declare.

scholarly journals MicroRNA-19b-3p promotes cell proliferation and osteogenic differentiation of BMSCs by interacting with lncRNA H19

2020 ◽  
Vol 21 (1) ◽  
Author(s):  
Gan Xiaoling ◽  
Liu Shuaibin ◽  
Liang Kailu
Keyword(s):  
Cell Proliferation ◽  
Protein Expression ◽  
Postmenopausal Osteoporosis ◽  
Critical Role ◽  
Dependent Manner ◽  
Cell Proliferation And Differentiation ◽  
Proliferation And Differentiation ◽  
17Β Estradiol ◽  
Dose Dependent

Abstract Background To investigated the role of miR-19b-3p in regulating bone marrow mesenchymal stem cell (BMSC) proliferation and osteoblast differentiation. Methods The expression of miR-19b-3p and lncRNA H19 were measured in postmenopausal osteoporosis patients and BMP-22 induced BMSCs using qRT-PCR. MiR-19b-3p mimic or inhibitor was transfected into BMP-2 induced BMSCs. Cell proliferation was measured by BrdU method. Protein expression of RUNX2 and COL1A1 were measured by western blot. PcDNA3.1-lncRNA H19 with or without miR-19b-3p mimic was transfected into BMP-2 induced BMSCs. Results The expression of miR-19b-3p was significantly up-regulated in postmenopausal osteoporosis patients and BMP-2 induced BMSCs. MiR-19b-3p overexpression dramatically elevated, while miR-19b-3p inhibition decreased cell proliferation of BMSCs. Additionally, protein expression levels of RUNX2 and COL1A1, as well as ALP activity were significantly promoted by miR-19b-3p mimic transfection and inhibited by miR-19b-3p inhibitor transfection. LncRNA H19 was obviously down-regulated in postmenopausal osteoporosis patients. H19 overexpression significantly decreased cell proliferation and differentiation by down-regulating miR-19b-3p. Moreover, the expression of miR-19b-3p was inhibited, while H19 elvated in 17β-estradiol (E2) treated BMSCs in a dose-dependent manner. Conclusion These data were the first to reveal the critical role of H19/miR-19b-3p in postmenopausal osteoporosis, and provided a new therapeutic target for OP.

CXCR4 Promotes the Tumorogenicity of Multiple Myeloma, Including Increased Motility, Clonogenicity, up-Regulation of VLA-4, Protection From Chemotherapy and Aggressive Tumor Development In Vivo

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1801-1801
Author(s):  
Katia Beider ◽  
Amnon Peled ◽  
Lola Weiss ◽  
Merav Leiba ◽  
Avichai Shimoni ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Molecular Mechanisms ◽  
Conflicts Of Interest ◽  
Critical Role ◽  
Tumor Development ◽  
Soft Agar ◽  
Induced Apoptosis

Abstract Abstract 1801 Background: Multiple myeloma (MM) is by large incurable neoplasm of plasma cells, characterized by accumulation in the bone marrow (BM), in close contact to cellular and extracellular matrix (ECM) components. Chemokine receptor CXCR4 is expressed by the majority of patients' MM cells. It promotes myeloma cell migration and homing to the BM compartment, supports the tumor cells survival and protects the myeloma cells from chemotherapy-induced apoptosis. Further investigation is required to define the specific molecular mechanisms regulated by the CXCR4/CXCL12 axis in MM. However, surface CXCR4 is commonly down-regulated in the MM cell lines. In order to overcome this limitation, the aim of the current study was to produce a reliable model for studying the functional role of high CXCR4 in MM by generating MM cell lines with stable expression of surface CXCR4. Results: To over-express CXCR4, we transduced CXCL12-expressing MM cell lines ARH77 and RPMI8226 with lentiviral vector and generated cell lines with high and stable levels of surface CXCR4. Enhanced CXCR4 expression significantly increased the in vitro survival and growth of the 2 MM cell lines in serum-deprivation conditions (p<0.01). Furthermore, elevated expression of surface CXCR4 prominently increased MM cells motility and promoted CXCL12-dependent transwell migration of the transduced MM cell lines. Highly CXCR4-expressing RPMI8226 and ARH77 cells demonstrated 40% migration in response to CXCL12 (50 ng/ml), versus only 0–5% migration of MM cells with low expression of surface CXCR4 (p<0.01). Furthermore, adhesion of MM cells to either ECM proteins or BMSCs localize the malignant PCs within the BM microenvironment, promote growth and survival of MM cells and play a critical role in myeloma bone disease and tumor invasion. In accordance, we observed induced adhesion of the transfected RPMI8226-CXCR4 cells to ECM components fibronectin and laminin and to BM fibroblasts. Moreover, we found that enhanced CXCR4 not only functionally activates, but rather significantly elevates the surface levels of VLA-4 integrin on the RPMI8226 cells. In addition, we found that CXCR4-expressing MM cells were less sensitive to melphalan- and bortezomib-induced apoptosis, when they were co-cultured with BM fibroblasts. Testing the molecular signaling pathways regulated by CXCR4, we found that elevated CXCR4 increased the basic level of pERK1/2 and pAKT in the MM cells, and promoted their prolonged activation in response to CXCL12 stimulation. Finally, the ability to produce colonies in the soft agar semi-solid culture reflects the tumorigenic capacity of cancer cells and cancer stem cells. Differentiated MM cells thus rarely produce colonies in soft agar. Here, we demonstrate that up regulation of CXCR4 promoted ARH77 and RPMI8226 colony formation, significantly increasing colonies number and size. Lastly, we determined the role of CXCR4 in MM tumor development in vivo. CXCR4-expressing ARH77 and RPMI8226 cells were subcutaneously injected into NOD/SCID mice. CXCR4-expressing cells, but not parental cell lines, produced detectable tumors already 10 days after the injection. Rapid tumor growth was further observed in both CXCR4-expressing cell lines. These findings indicate that CXCR4 provided aggressive phenotype and supported MM growth in vivo. Conclusions: Taken together, our findings clearly demonstrate the important pathophysiologic role of CXCR4 in MM development and progression. Furthermore, for the first time, we provide the evidence for CXCR4 oncogenic potential in MM, showing that CXCR4 promotes the clonogenic growth of MM cells. Our model may further serve to elucidate CXCR4-regulated molecular events potentially involved in the pathogenesis of MM, and strongly support targeting CXCR4 as therapeutic tool in MM. Disclosures: No relevant conflicts of interest to declare.

Mir-29a Displays in Vitro and in Vivo Anti-Tumor Activity in Multiple Myeloma

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2090-2090
Author(s):  
Manujendra N Saha ◽  
Yan Chen ◽  
Jahangir Abdi ◽  
Hong Chang
Keyword(s):  
Multiple Myeloma ◽  
Tumor Suppressor ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Therapeutic Potential ◽  
Critical Role ◽  
Treatment Strategies ◽  
Loss Of Function

Abstract Despite advances in recent therapeutic approaches including targeted therapies, multiple myeloma (MM) remains still incurable necessitating the development of novel treatment strategies. MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate post-transcriptional gene expression and play a critical role in tumor pathogenesis. Tumor suppressor miRNAs are generally down-regulated in cancer cells compared to their normal counterpart, and their enforced expression indeed represents a promising strategy for cancer treatment. In this study, we sought to characterize the role of miR-29a as a tumor suppressor as well as evaluated its therapeutic potential in MM. miR-29a expression levels were found down-regulated in a panel of 5 MM cell lines, 6 newly diagnosed MM patient samples compared to its expression in normal hematopoietic cells collected from 10 normal healthy individuals suggesting that high expression of miR-29a might be involved in MM pathogenesis. We further assessed the functional significance of miR-29a by both gain- and loss-of-function studies. A significant decrease in cell viability (22-32%, p<0.05), along with induction of apoptosis (30-35%, p<0.05) was observed at 48 hrs in MM cell lines, MM.1S and 8226 transfected with miR-29a compared to cells transfected with scrambled miRNA. In contrast, cell lines transfected with miR-29a antagonist prevented the loss of viability in such cells indicating the specificity of miR-29a. At the molecular level, we have identified c-Myc, an important oncogenic transcription factor known to stimulate MM cell proliferation, as a target of miR-29a. Binding site of miR-29a was first identified by computer algorithm and further confirmed by the use of a 3’UTR of c-Myc reporter (luciferase renilla/firefly) constructs containing, miR-29a target site. Moreover, treatment with PRIMA-Met, a small molecule anti-tumor agent in phase I/II clinical trials, significantly increased the expression of miR-29a (2 to 6-fold) and decreased expression of c-Myc in MM cell lines and primay MM patient samples suggesting an important role of miR-29a in inhibiting proliferation of MM cells. On the other hand, overexpression of c-Myc in 8226 and MM.1S cells at least partially reverted the functional effect of miR-29a or PRIMA-1Metsuggesting a specific role of c-Myc in mediating its anti-proliferative activity. To examine therapeutic potential of our studies, we took advantage of novel lipid based delivery method of miRNA. Intratumor delivery of the miR-29a by intraperitoneal injection route against MM xenografts in SCID mice resulted in a significant inhibition of tumor growth (~60%) at 12 days of treatment and prolongation of survival (median survival increased from 22 days to 35 days, p<0.038) compared to the mice receiving scrambled miRNA. Retrieved tumors from treated mice showed efficient increase in miR-29a (5.5-fold, p=0.025), and decrease in c-Myc protein as well as reduced expression of Ki67 and increase of Tunel expression. Similar phenomenon was observed by systematic delivery of miR-29a (by intraveneous injection) in mice with no significant side effects or toxicity in mice. Our study reveals an important role of miR-29a as a tumor suppressor in mediating anti-tumor activities in MM cells by targeting c-Myc. Our findings provide a proof-of-principle that formulated synthetic miR-29a exerts therapeutic activity in preclinical models, and support a framework for development of miR-29a based treatment strategies in MM patients. Disclosures No relevant conflicts of interest to declare.

scholarly journals HDAC8 Maintain Cytoskeleton Integrity Via Homologous Recombination and Represent a Novel Therapeutic Target in Multiple Myeloma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4385-4385
Author(s):  
Zuzana Chyra ◽  
Maria Gkotzamanidou ◽  
Masood A. Shammas ◽  
Vassilis L. Souliotis ◽  
Yan Xu ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Dna Repair ◽  
Homologous Recombination ◽  
Cell Lines ◽  
Hdac Inhibitors ◽  
Mass Spectrometry Analysis ◽  
Dependent Manner ◽  
Large Panel ◽  
Dose Dependent

Multiple Myeloma (MM) is a plasma cell malignancy vulnerable to epigenetic intervention, with histone deacetylases (HDACs) emerging as the most promising epigenetic targets in combination with current anti-myeloma agents. Pan-HDAC inhibitors are effective as therapeutic agents both in preclinical and clinical setting; however, there is an increasing emphasis on understanding the biological and molecular roles of individual HDACs to limit toxicities observed with pan-HDAC inhibitors. Based on correlation with patient outcome in three independent myeloma datasets, we have evaluated the functional role of HDAC8, a member of Class I HDAC isoenzymes, in MM. Unlike other isoforms, there is limited information about molecular and epigenomic functions of HDAC8. We have previously confirmed expression of HDAC8 in a large panel of MM cell lines, where it is localized predominantly to cytoplasm. Moreover, genetic and pharmacological modulation of HDAC8 with RNAi and specific inhibitor PCI-34051 resulted in a significant inhibition of myeloma cell proliferation and decrease in colony formation (p<.001). HDAC8 inhibition led to an increase in the ongoing spontaneous and radiation-induced DNA damage in MM cells by affecting DNA repair via the homologous recombination (HR) pathway, suggesting a novel function of HDAC8 in promoting HR and DNA repair in MM cells. Using laser micro-irradiation in MM1S and U2OS cells, we observed HDAC8 recruitment to DSBs sites and its co-localization with Rad51 and Scm3, a member of cohesin complex. A transcriptomic analysis of HDAC8 knock-down cells also shows perturbation of number of cytoskeleton-related genes confirming significant role of HAD8 in cytoskeleton rearrangement in MM. Mass-spectrometry analysis to identify the HDAC8 substrates in MM cells is currently ongoing. Classical pan-HDACi, such as SAHA (vorinostat), bind to HDAC8 with substantially diminished activity (IC50 = 2 μM), reflecting a unique binding site of this isoform. To discover and validate new small molecules with HDAC8 subtype selectivity, we have explored the efficacy of OJI-1, a novel selective and potent HDAC8 inhibitor (IC50 = 0.8 nM) with modest inhibition of HDAC6 (1200 nM). Treatment with OJI-1 selectively impact cell viability of a large panel of MM cell lines (n=20) in a time and dose dependent manner, while sparing healthy donors PBMC both in resting and activated state (n=3). The significantly higher IC50 observed in PBMCs suggests a favorable therapeutic index. Western blotting analysis confirmed target selectivity with significant time and dose dependent decrease in H3 and H4 acetylation in MM cells treated with OJI-1. Moreover, pharmacological inhibition of HDAC8 specifically inhibited HR but not non-homologous end joining. These data suggest that targeting of HDAC8 using OJI-1 could be effective treatment approach in MM. Based on molecular data combination studies and in vivo evaluation are ongoing. In conclusion, our results provide insight into the role of HDAC8 in DNA stability and cell growth and viability which can be exploited in future for therapeutic application alone and in combination in MM. Disclosures Munshi: Takeda: Consultancy; Janssen: Consultancy; Amgen: Consultancy; Abbvie: Consultancy; Janssen: Consultancy; Celgene: Consultancy; Takeda: Consultancy; Adaptive: Consultancy; Amgen: Consultancy; Adaptive: Consultancy; Abbvie: Consultancy; Oncopep: Consultancy; Oncopep: Consultancy; Celgene: Consultancy.

scholarly journals MDMX is essential for the regulation of p53 protein levels in the absence of a functional MDM2 C-terminal tail

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Jack D. Sanford ◽  
Jing Yang ◽  
Jing Han ◽  
Laura A. Tollini ◽  
Aiwen Jin ◽  
...  
Keyword(s):  
Dna Damage ◽  
Protein Expression ◽  
P53 Protein ◽  
Critical Role ◽  
E3 Ligase ◽  
Mouse Embryonic Fibroblast ◽  
Protein Levels ◽  
P53 Protein Expression ◽  
C Terminus

Abstract Background MDM2 is an E3 ubiquitin ligase that is able to ubiquitinate p53, targeting it for proteasomal degradation. Its homologue MDMX does not have innate E3 activity, but is able to dimerize with MDM2. Although mouse models have demonstrated both MDM2 and MDMX are individually essential for p53 regulation, the significance of MDM2-MDMX heterodimerization is only partially understood and sometimes controversial. MDM2C462A mice, where the C462A mutation abolishes MDM2 E3 ligase activity as well as its ability to dimerize with MDMX, die during embryogenesis. In contrast, the MDM2Y487A mice, where the Y487A mutation at MDM2 C-terminus significantly reduces its E3 ligase activity without disrupting MDM2-MDMX binding, survive normally even though p53 is expressed to high levels. This indicates that the MDM2-MDMX heterodimerization plays a critical role in the regulation of p53. However, it remains unclear whether MDMX is essential for the regulation of p53 protein levels in the context of an endogenous MDM2 C-terminal tail mutation. Results Here, we studied the significance of MDM2-MDMX binding in an MDM2 E3 ligase deficient context using the MDM2Y487A mouse embryonic fibroblast (MEF) cells. Surprisingly, down-regulation of MDMX in MDM2Y487A MEFs resulted in a significant increase of p53 protein levels. Conversely, ectopic overexpression of MDMX reduced p53 protein levels in MDM2Y487A MEFs. Mutations of the RING domain of MDMX prevented MDMX-MDM2 binding, and ablated MDMX-mediated suppression of p53 protein expression. Additionally, DNA damage treatment and nuclear sequestration of MDMX inhibited MDMX activity to suppress p53 protein expression. Conclusions These results suggest that MDMX plays a key role in suppressing p53 protein expression in the absence of normal MDM2 E3 ligase activity. We found that the ability of MDMX to suppress p53 levels requires MDM2 binding and its cytoplasmic localization, and this ability is abrogated by DNA damage. Hence, MDMX is essential for the regulation of p53 protein levels in the context of an MDM2 C-terminal mutation that disrupts its E3 ligase activity but not MDMX binding. Our study is the first to examine the role of MDMX in the regulation of p53 in the context of endogenous MDM2 C-terminal mutant MEF cells.

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