ANKHD1 Silencing Reduces In Vitro Clonogenicity and In Vivo Tumorogenicity Of Multiple Myeloma Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3168-3168
Author(s):  
Anamika Dhyani ◽  
João Agostinho Machado-Neto ◽  
Patricia Favaro ◽  
Sara Teresinha Olalla Saad
Keyword(s):  
Cell Cycle ◽  
Gene Therapy ◽  
Multiple Myeloma ◽  
Cell Lines ◽  
Tumor Size ◽  
Control Group ◽  
Mice Model ◽  
And Migration

Abstract Introduction ANKHD1 is a multiple ankyrin repeats containing protein, highly expressed in cancers, such as acute leukemia. Earlier studies showed that ANKHD1 is highly expressed and plays important role in proliferation and cell cycle progression of multiple myeloma (MM) cells. It was also observed that ANKHD1 downregulation modulates cell cycle gene expression and upregulates p21 irresepective of TP53 mutational status of MM cell lines. Objective The present study aimed to study the effect ofANKHD1 silencing on MM growth both in vitro (clonogenicity, migration) and in vivo (xenograft tumor mice model). The purpose was to investigate the feasibility of ANKHD1 gene therapy for MM. Methods In the present study, ANKHD1 expression was silenced using short hairpin RNA (shRNA)-lentiviral delivery vector in MM cell lines (U266 and MM1S). For control MM cells were tranduced by lentiviral shRNA against LacZ. Downregulation of ANKHD1 expression was confirmed by qPCR and Western blot. Colony formation capacity and migration of control and ANKHD1 silenced MM cells was determined by methylcellulose and transwell migration assays, respectively. For in vivo MM growth, NOD-SCID mice were divided in two groups injected with control and ANKHD1 silenced cells, separately. Mice were observed daily for tumor growth. Once the tumor size reached 1 mm3, mice in both groups were sacrificed and tumor was excised to measure tumor volume and weight. Results Corroborating the results obtained in our earlier studies, in the present study also inhibition of ANKHD1 expression suppressed growth of MM cells in vitro. MM cell lines tranduced with ANKHD1 shRNA showed significantly low number of colonies ten days after plating in methylcellulose medium as compared to control (p<0.05). Similarly, in transwell migration assay, cell lines transduced with ANKHD1 showed significantly less migration as in response to 10% FBS at lower chamber as compared to control group (p<0.05) in both the cell lines analyzed. Further in xenograft MM mice model, the growth of tumor was visibly suppressed in mice injected with ANKHD1 silenced cells compared to control group. There was significant difference in tumor size (volume) between these 2 groups (P< 0.006). The tumor weight of the inhibition group was 0.71 ±0.2 g, significantly lighter than those of the control group (1.211 ± 0.5 g, P =0.02) Conclusion Our data indicates ANKHD1 downregulation significantly inhibits colony-forming ability and migration of both glucocorticoid resistant (U266) and sensitive (MM1S) MM cells. Further, gene silencing of ANKHD1 also resulted in reduced in vivo tumor growth in NOD/SCID mice. Collectively, the result obtained indicates that ANKHD1 may be a target for gene therapy in MM. Disclosures: No relevant conflicts of interest to declare.

The Dual PIM/PI3-K Inhibitor Ibl-202 Overcomes Microenvironmental Mediated Resistance in Multiple Myeloma and Prevents PIM1 Induced CXCR4 Upregulation

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 5350-5350
Author(s):  
Mairead Reidy ◽  
Marianne VanDijk ◽  
Michael O'Neill ◽  
Michael O'Dwyer
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Lines ◽  
Surface Expression ◽  
Down Regulation ◽  
Pim Kinases ◽  
Ic50 Values ◽  
And Migration

Abstract Background: The interaction of multiple myeloma (MM) cells with bone marrow (BM) cells along with factors in the BM milieu such as chemokines and cytokines play a crucial role in both progression of MM and drug resistance. Activation of the PI3-K/Akt survival pathway is a characteristic of both human MM cell lines and patient samples. This activation can be linked to BM microenvironmental signalling and use of proteasome inhibitors in treatment, suggesting this as a crucial point of therapeutic intervention to abrogate growth and survival signals in MM. However, the efficacy of such therapeutics has been modest and is likely to be compromised by the stimulation of compensatory signalling pathways, such as the PIM kinases, which like the PI3-K/Akt pathway are also induced by BM microenvironmental influences and share similar downstream targets. These proto-oncogenic kinases are constitutively active and play an important role in proliferation and survival in MM. The influence of these kinases on homing and migration has been observed in other malignancies, this has yet to be reported in MM. Here we report the effects of a dual inhibitor of PIM/PI3-K, IBL-202, and provide novel insights into effects on cell survival, signaling and migration. Methods: We investigated the effect of IBL-202 against a panel of MM cell lines (MM.IS, NCI-H929s, KMS11 and RPMI-8226) and primary MM patient samples. The in vitro efficacy of IBL-202 was compared to that of single pan-PIM inhibitors pPIMi and AZD1208 and also the pan-PI3-K inhibitor GDC-0941. Apoptosis was measured with AnnexinV staining and cell cycle analysed with Edu/DAPI staining. To mimic BM microenvironmental conditions MM cells were cultured under hypoxic conditions (1% O2) and in co-culture with the human stromal cell line HS5. Surface expression of CXCR4 was assessed in MM cell lines by flow cytometry. PIM kinases, pCXCR4 and downstream targets of PIM/PI3-K were examined by western blot. Transwell migration assays were carried out in the presence of 50ng SDF-1α for 4h @ 37o C. Results: Simultaneous inhibition of PIM and PI3-K using IBL-202 in vitro was significantly more potent at inducing apoptosis than GDC-0941, pPIMi or AZD1208 in all MM cell lines tested. IC50 values were under 1μM for IBL-202 at 48h whilst in comparison the pan PIM inhibitors pPIMi and AZD1208 scored IC50 values between 5 and 10μM. The IC50 for GDC-0941 was on average 5μM (Figure 1). At the molecular level there was a notable decrease in phosphorylation of known PIM/PI3-K targets Akt (Ser473), Bad (Ser112) and the translational targets S6 (Ser235/236) and 4EBP1 (Thr37/46). The levels of total proteins were unchanged. Treatment with increasing doses of IBL-202 led to a marked reduction in cells in S phase of the cell cycle. These changes were paralled by down regulation of the cell cycle promoting proteins cyclin D1 and c-myc. IBL-202 was also effective in inducing apoptosis in primary MM patient samples (n=4) after just 24h as assessed by Annexin-V staining (Figure 2). To explore the role of the BM microenvironment we co-cultured MM cell lines with HS5s. This led to strong induction of PIM2 in MM cells. While MM cells in this setting were protected from Bortezomib-induced cell death, the apoptotic effect of IBL-202 was enhanced. In a further effort to mimic the tumour microenvironment we cultured MM cell lines in hypoxia. This may be of particular relevance as Pim-1 has been reported to be a pivotal regulator involved in hypoxia-induced chemoresistance. MM cells were further sensitised to IBL-202 in hypoxia. In addition, hypoxia increased the surface expression of CXCR4, a chemokine receptor critical for homing of MM cells to the bone marrow, with a concomitant increase in PIM1. Treatment of MM cell lines with IBL-202 reduced the level of PIM1 and CXCR4 Ser339 phosphorylation, along with down regulation of CXCR4 surface expression resulting in reduced migration of MM cells along an SDF-1 gradient. Conclusion: Together these data provide direct evidence of the potency of IBL-202 in MM in conditions that mimic the BM microenvironment. Moreover, they indicate a potential role for PIM kinases in facilitating dissemination and invasiveness of MM by CXCR4 and provide an added rationale for targeting PIM kinases in MM. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures O'Neill: Inflection Biosciences: Employment.

scholarly journals Novel Combination Therapy Targeting rDNA Transcription and Histone Deacetylation Provides Effective Treatment for Multiple Myeloma, and Synergises in Bortezomib-Resistant MM

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1945-1945
Author(s):  
Kylee H Maclachlan ◽  
Andrew Cuddihy ◽  
Nadine Hein ◽  
Carleen Cullinane ◽  
Simon J. Harrison ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Drug Resistance ◽  
Cell Lines ◽  
Cellular Response ◽  
Single Agent ◽  
Resistant Cell ◽  
Rdna Transcription

Abstract Background: Multiple myeloma (MM) requires combination drug therapies to delay acquired drug resistance and clinical relapse. We co-developed CX-5461, a highly-selective inhibitor of RNA polymerase I-mediated rDNA transcription(1), currently in phase I trials for relapsed haematological malignancies (Peter Mac). CX-5461 produces a targeted nucleolar DNA damage response (DDR), triggering both a p53-dependent and -independent nucleolar stress response and killing malignant cells while sparing normal cells(2,3). Single-agent CX-5461 provides an impressive survival benefit in mouse models of B-cell lymphoma, acute myeloid leukaemia and now MM(2,4,5). However, drug resistance eventually occurs, confirming the need for combination therapies. Aim: To test the efficacy of CX-5461 in combination with the histone deacetylase inhibitor panobinostat, (prioritised from a boutique high-throughput screen of anti-myeloma agents), with a focus on the setting of resistance to proteasome-inhibitors (PIs). Methods: We assessed the impact of CX-5461 and panobinostat on overall survival in mouse models of MM, then surveyed the effects on cellular response and molecular markers of DDR. We developed bortezomib-resistant cell lines and an in vivo model of bortezomib-resistance to test this combination in the setting of PI-resistance. Results: CX-5461 in combination with panobinostat provides a significant survival advantage in both the transplanted Vk*MYC and the 5T33/KaLwRij models, with minimal bone marrow toxicity. The combination showed increased anti-proliferative effects and cell death in vitro. Interestingly, experiments interrogating the downstream cellular response of this combination suggest that the mechanism(s) driving synergy are complex and cell context-dependent. Cell cycle analysis indicates that both CX-5461- and panobinostat-driven cell cycle effects, i.e. G2/M and G1/S arrest, respectively, are dominant in the combination setting in a cell line-dependent manner, suggesting that context-dependent factors such as p53 may influence the cellular response. Mechanistically, in both p53-wild type and -null cell lines we observe an increase in DDR signalling with single agent CX-5461, with only moderate further increase with the combination. Moreover, CX-5461-mediated MYC downregulation is not universally observed, with the combination promoting further downregulation only in some cell lines. Given the potential for affecting global transcription programs downstream of panobinostat, we are performing transcriptome analyses in the combination setting compared to single agent treatment. We have generated bortezomib-resistant cell lines, sequentially increasing drug exposure to establish populations growing at concentrations above the IC90 of the parental lines. The resistant 5T33 cells retain their resistance to bortezomib in vivo and we have demonstrated that CX-5461 remains effective in this model, significantly increasing survival. We are currently examining the combination of CX-5461 with panobinostat in this model of bortezomib-resistance, which will give critical information guiding patient selection for future clinical trials. Conclusion: The rDNA transcription inhibitor CX-5461 synergises in vitro and in vivo with panobinostat, and CX-5461 retains efficacy in the setting of bortezomib-resistant myeloma. References Drygin et al., Cancer Research 2011 Bywater et al., Cancer Cell 2012 Quin et al, Oncotarget, 2016 Devlin et al., Cancer Discovery 2016 Hein et al., Blood 2017 Disclosures Harrison: Janssen-Cilag: Other: Scientific advisory board.

scholarly journals The Role of PHF19 As a Promoter of Tumorigenicity and Therapeutic Target in Multiple Myeloma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 508-508
Author(s):  
Carolina D. Schinke ◽  
Pingping Qu ◽  
Shmuel Yaccoby ◽  
Valeriy V Lyzogubov ◽  
Veronica MacLeod ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Candidate Gene ◽  
Cell Lines ◽  
Crucial Role ◽  
Plasma Cells ◽  
Total Therapy

Introduction - Multiple Myeloma (MM) is a hematologic malignancy characterized by clonal growth of differentiated plasma cells (PCs). Despite improvement in MM therapy, the disease remains mostly incurable and is characterized by recurrent relapses with development of resistant clones that eventually lead to patient death. The pathways that lead to resistant and aggressive MM are not fully understood highlighting the need to improve our understanding of MM biology to identify potential new pathways and therapeutical targets. PHD Finger Protein 19 (PHF19) is a regulator of Polycomb Repressive Complex 2 (PRC2), the sole methyltransferase complex capable of catalyzing H3K27me3 to induce and enforce gene repression. PRC2 employs enhancer of zeste homolog 1 and 2 (EZH1/EZH2) as enzymatic subunits to hypermethylate H3K27. While overexpression and gain of function mutations of EZH1/2 have been observed in many cancers the role of this particular pathway in MM remains poorly understood. In the present study, we report on PHF19 as a new candidate gene to play a potential crucial role in MM oncogenesis. Methods- Gene expression profiling (GEP; Affymetrix U133 Plus 2.0) was performed on 739 MM patients (from total therapy trials [TT] 3-5; low risk MM n=636, high risk MM n=103), 42 patients with monoclonal gammopathy of undetermined significance (MGUS), 73 smoldering MM patients, 42 patients with primary plasma cell leukemia and 34 healthy donors. Myeloma risk was determined by the GEP 70 signature as previously defined. To test the implications of functional PHF19 knock down (KD) we used TRIPZ inducible PHF19 shRNA vs. scrambled control (Dharmacon) in two MM cell lines (JJN3 and ARP1). Real time PCR as well as western blotting was used to confirm PHF19 KD as well as to elucidate the effect on H3K27me3 (Cell Signaling). Functional in vitro studies included proliferation (Promega), clonogenic assays (StemCell), cell cycle and apoptosis assays (both Invitrogen). In vivo studies were performed using SCID mice that were subjected to tail vain injection with PHF19 KD JJN3 cells (n=10) or scrambled shRNA control (n=10). Weekly ELISA (Bethyl) and in vivo imaging (Xenogen) were performed and survival was recorded. Results- GEP of the previously mentioned patient populations and healthy controls identified PHF19 (chr9q33.2) as a candidate gene that was consistently dysregulated in MM patients. Mean expression levels at different MM stages correlated with disease aggressiveness (ANOVA, p&lt;0.0001), Figure 1. High expression of PHF19 (log2&gt;10.46) at diagnosis correlated significantly with adverse clinical parameters, including ISS III, anemia and elevated LDH, as well as worse overall survival (5 yr OS = 29% for patients with high PHF19 expression vs 77% for patients with low PHF19 expression [log2&lt;10.46], p&lt; 0.0001). These results led us to test the implications of functional PHF19 KD using TRIPZ inducible PHF19 shRNA vs. scrambled control in the JJN3 and ARP1 MM cell lines. PHF19 KD led to a drastic reduction of H3K27me3 thereby resulting in significantly reduced proliferation via cell cycle arrest, while apoptosis was not substantially altered. Clonogenic assays showed a significant reduction in colony numbers and size of MM cells with PHF19 KD compared to the control (&gt;75% reduction in both cell lines, p&lt;0.05). Xenograft studies showed consistently less tumor burden in the mice injected with PHF19 KD cells compared to scrambled control, evident through ELISA testing for IgG Kappa (Median =180 mg/ml for scrambled control vs 80 mg/ml for PHF19 KD at week 8, p=0.07) and bioimaging (Median bioilumisence 2.1x108 p/s for scrambled control vs. 0.8x108 p/s for PHF19 KD at week 8, non-significant). Median OS in mice injected with PHF19 KD cell was substantially longer (66 days) compared to mice subjected to scrambled control cells (54 days), p=0.052. Conclusion- In summary we show that PHF19 is upregulated in malignant plasma cells of MM patients and that PHF19 expression levels increase with advanced MM stages. High PHF19 expression was a marker of adverse prognosis in our total therapy (TT 3-5) cohort. Most importantly, in-vitro and in-vivo functional studies showed that PHF19 has important biological functions in MM. These results suggest that epigenetic regulation through histone methylation, in particular, H3K27 trimethylation, plays a crucial role in MM and the affected downstream pathways should be further elucidated. Disclosures Boyle: Janssen: Honoraria, Other: Travel; Abbvie: Honoraria; Amgen: Honoraria, Other: travel; Takeda: Honoraria, Other: travel; Celgene Corporation: Honoraria, Other: Travel. van Rhee:Kite Pharma: Consultancy; Adicet Bio: Consultancy; Karyopharm Therapeutics: Consultancy; Takeda: Consultancy; Sanofi Genzyme: Consultancy; Castleman Disease Collaborative Network: Consultancy; EUSA: Consultancy. Walker:Celgene: Research Funding.

scholarly journals Glaucocalyxin A suppresses multiple myeloma progression in vitro and in vivo through inhibiting the activation of STAT3 signaling pathway

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Mei Li ◽  
Cailong Chen ◽  
Qian Wang ◽  
Xiaolu Jiang ◽  
Lanlan Tan ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Cell Proliferation ◽  
Xenograft Model ◽  
Mice Model ◽  
Cycle Arrest ◽  
Nude Mice Model

Abstract Background Multiple myeloma (MM) is the most common malignant hematological disease in the people worldwide. Glaucocalyxin A (GLA) is a bioactive ent-kauranoid diterpenoid, that is derived from Rabdosia japonica var. GLA has been demonstrated that it had various pharmacological activities, such as anti-coagulation, anti-bacterial, anti-tumor, anti-inflammation, antioxidant activities. Although GLA has effective anti-tumor properties, its effects on multiple myeloma remain unclear. The aim of this study was to examine the possible anti-cancer effects of GLA and their molecular mechanisms on MM cells in vitro and in vivo. Methods To evaluate the role of GLA on the proliferation of MM cells in vitro and in vivo, we used MTT method to detect the role of GLA on the proliferation of MM cells. Cell apoptosis and cell cycle assay were evaluated by flow cytometry. Protein expressions in GLA-treated and untreated MM cells were evaluated by western blot analyses. MM xenograft nude mice model was used to investigate the role of GLA on the proliferation of MM cells in vivo. IHC assay was used to examine the role of GLA on the MM xenograft model in vivo. Results In the present study, we firstly reported the potent anti-myeloma activity of GLA on MM cells. We found that GLA could induce apoptosis in vitro and in vivo. GLA could inhibit the phosphorylation of the signal transducer and activator of transcription 3 (STAT3) and downregulate interleukin IL-6 induced STAT3 phosphorylation in MM. Overexpression of STAT3 could significantly prevent apoptosis induced by GLA; while knockdown of STAT3 enhanced it. Moreover, GLA could inhibit cell proliferation by inducing the cell cycle arrest. GLA reduced the expression of cell cycle-related proteins CCNB1, CCND1, CCND2, and CCND3 and increased the expression of p21 in MM cell lines. In addition, in the MM xenograft nude mice model, GLA exhibited very good anti-myeloma activity. Administration of GLA almost completely inhibited tumor growth within 19 days without physical toxicity. And the IHC results showed GLA significantly inhibited cell proliferation and interfered STAT3 pathway on MM xenograft model tumor tissues. Conclusions Taken together, our present research indicated that GLA inhibits the MM cell proliferation, induces MM cell apoptosis and cell cycle arrest through blocking the activation of STAT3 pathway. Thus, GLA may be a potential therapeutic candidate for MM patients in the future.

Novel PI3K Inhibitor Compound A Induces Myeloma Cell Apoptosis and Shows Synergistic Cytotoxicity with Dexamethasone In Multiple Myeloma

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4080-4080
Author(s):  
Yuhuan Zheng ◽  
Jing Yang ◽  
Liang Zhang ◽  
Jianfei Qian ◽  
Jairo Matthews ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Cell Growth ◽  
Western Blot ◽  
Cell Lines ◽  
Blot Analysis ◽  
Cell Apoptosis ◽  
Compound A

Abstract Abstract 4080 Phosphatidylinositol 3-kinase (PI3K) plays a central role in cell metabolism. PI3K is activated by growth factors, cytokines, and other stimulatory factors in association with their receptors. Activated PI3K in turn initiates signaling transduction to Akt-mTOR and leads to regulation of cell growth, proliferation, and apoptosis. Dysregulation of the pathway is widely seen in different types of human cancers, including multiple myeloma (MM). Therefore, PI3K-Akt inhibition is expected to exert broad anti-MM activity. Compound A (CA) is a novel pan-PI3K inhibitor, developed by Novartis Oncology. This compound has shown significant cell growth inhibition and induction of apoptosis in a variety of tumor cell lines. CA is currently being investigated in Phase I clinical trials in solid tumor patients. In this study, we investigated the in vitro and in vivo anti-MM activity of CA. Our findings showed that CA induces apoptosis in MM cell lines, ARP1, ARK, MM.1S, MM.1R, CAG and U266, and primary MM cells in both a time-dependent and a dose-dependent manner in vitro. Western blot analysis indicated activation of caspases after CA exposure. The presence of MM bone marrow stromal cells (BMSCs) or addition of IL-6, the growth cytokine for MM, did not attenuate CA-induced MM cell apoptosis. More importantly, CA only showed limited cytotoxicity toward normal lymphocytes or non-tumoric BMSCs. Results from mechanistic studies showed that CA treatment results in cell cycle arrest in G1 phase by upregulating cell cycle repressor p27 (Kip1) and downregulating cyclin D1. CA treatment also caused decreased anti-apoptotic XIAP expression, and increased cytotoxic small isoform of Bim, BimS expression, both of which may contribute to CA-induced cell apoptosis. In addition to its effect in vitro, CA showed potent anti-MM activity in vivo in an established MM model in SCID mice. CA treatment repressed tumor growth and prolonged the survival of tumor-bearing mice. To test the synergistic/addictive effect of CA with other MM chemotherapeutics, we combined CA with melphalan, dexamethasone, lenalidomide, or bortezomib to treat MM cells. Our results showed that low doses of CA and dexamethasone, either of which alone has only limited cytotoxicity, exhibited synergistic anti-MM activity in dexamethasone-sensitive cell lines ARP1 and MM.1S, but not in dexamethasone-resistant cell MM.1R. Western blot analysis suggested that CA and dexamethasone combined treatment in MM.1S results in accumulation of the cytotoxic BimS. Increased BimS expression may cause the synergistic effect of CA and dexamethasone. Thus, our findings suggest CA alone or together with dexamethasone may be a promising treatment for MM. Disclosures: No relevant conflicts of interest to declare.

scholarly journals JAM-A as a Prognostic Factor and New Therapeutic Target in Multiple Myeloma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 307-307 ◽  
Author(s):  
Antonio Solimando ◽  
Andreas Brandl ◽  
Mattenheimer Katharina ◽  
Carolin Graf ◽  
Miriram Ritz ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Therapeutic Target ◽  
Healthy Subjects ◽  
Plasma Cells ◽  
Cell Membrane Protein ◽  
And Migration ◽  
Rpmi 8226

Abstract Cell adhesion in the multiple myeloma (MM) microenvironment is a mechanism by which MM plasma cells escape the effects of therapy and survive. To improve clinical strategies and overcome drug resistance, approaches directed to both MMPCs and bone marrow microenvironment are under investigation. Here, we examined the cell membrane protein Junctional adhesion molecule-A (JAM-A) as a clinical biomarker and novel therapeutic target for MM. We evaluated JAM-A expression by real time PCR (RT-PCR), flow cytometry and immunofluorescence microscopy in 132 MM patients at different stages and various MM cell lines. Next, we measured the concentrations of soluble JAM-A from MM and healthy subjects sera by enzyme linked immune assay (ELISA). We investigated JAM-A functionally in vitro and in vivo by transient gene silencing (siRNA) and with blocking antibodies. Patient-derived plasma cells (MMPCs) expressed increased JAM-A expression levels when compared to control PC from healthy individuals. Elevated JAM-A expression correlated with poor prognosis (Figure 1A,B). Furthermore, soluble JAM-A was significantly increased in MM patient sera when compared to healthy subjects. Additionally, MM cell lines showed high expression of both membrane and cytoplasmic JAM-A. Consequently, inhibition of JAM-A using specific siRNA treatment resulted in diminished tumorigenic potential, including decreased colony formation, chemotaxis and migration. Importantly, treatment of luciferase+RPMI-8226 MM bearing NSG with a JAM-A blocking monoclonal antibody reduced significantly MM progression and dissemination in vivo when compared to MM bearing mice that received an non-specific isotype control antibody (Figure 1C). Conclusively, our data suggest that JAM-A can serve as a biomarker of malignancy in MM patients. Soluble plasma JAM-A could contribute to serum-based clinical stratification. Furthermore, therapeutic targeting of JAM-A appears attractive for clinical translation. Figure 1 Figure 1. Disclosures Einsele: Celgene: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; Amgen: Consultancy, Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria.

Hypoxia Induces Pgp-Mediated Carfilzomib Resistance in Multiple Myeloma Cells and HIF Inhibition Significantly Enhances Sensitivity and Response to Carfilzomib In Vivo

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1286-1286
Author(s):  
Barbara Muz ◽  
Joseph Abraham ◽  
Feda Azab ◽  
Pilar De La Puente ◽  
Nicholas Potter ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Tumor Progression ◽  
Cell Lines ◽  
Induced Resistance ◽  
Tumor Size ◽  
Research Funding ◽  
Scid Mice ◽  
Tumor Initiation

Abstract Introduction: A plasma cell malignancy called multiple myeloma (MM) is an incurable cancer in which a majority of patients with refractory disease become resistant to therapy. The introduction of carfilzomib, a proteasome inhibitor, significantly improved the clinical outcome of the MM treatment. However, MM patients develop resistance to carfilzomib and relapse. In this study, we investigated the role of hypoxia and P-gp in the carfilzomib-resistance in MM cells in vitro and in vivo, and its therapeutic translational potential using a HIF inhibitor, PX-478. Methods: In vitro, MM cell lines were treated with carfilzomib under normoxic (21% O2) or hypoxic (1% O2) conditions and cell survival was analyzed by MTT assay. The activity of P-gp was assessed by testing the efflux of a known P-gp substrate, RhodamineB (RhoB), in the hypoxic and normoxic conditions by measuring the intracellular RhoB content using flow cytometry. Moreover, we have tested the effect of HIF inhibition using PX-478 on the P-gp activity as well as the response to carfilzomib in hypoxic and normoxic cells. In vivo, we tested the effect of HIF inhibition on tumor initiation, where MM1s-Luc-GFP cells were injected intravenously (IV) into SCID mice, which were treated instantaneously with PX-478 (10mg/kg) three times a week. Furthermore, we tested the effect of PX-478 on MM tumor response to carfilzomib (measuring tumor size and mice survival). MM1s-Luc-GFP cells were injected IV into SCID mice, tumors grew for 3 weeks and the mice were then randomly divided into 4 groups treated with (1) vehicle (Captisol), (2) PX-478 (10mg/kg) alone, (3) carfilzomib (5mg/kg) alone, or (4) a combination of PX-478 (10mg/kg) and carfilzomib (5mg/kg). PX-478 or vehicle were administered by oral gavage three times a week (day 1, day 3, day 5), while vehicle and carfilzomib were injected IV twice a week (day 4 and day 5). Tumor size was imaged using bioluminescence imaging (BLI) and mice survival was followed for 70 days. Results: In vitro, we found that hypoxia induced resistance to carfilzomib in five MM cell lines. Moreover, hypoxia also increased activity of P-gp by causing decreased intracellular RhoB content in hypoxic MM cells. The HIF inhibitor, PX-478, as well as the P-gp inhibitor, tariquidar, reversed the activation of P-gp in hypoxic cells, while the combination of PX-478 and tariquidar did not induce further inhibition of P-gp activity. Furthermore, the combination of PX-478 or tariquidar with carfilzomib reversed the hypoxia-induced resistance in MM. However, tariquidar and other P-gp inhibitors have shown low selectivity and high toxicity in clinical trials; therefore, for our in vivo experiments we chose to inhibit HIF in order to reverse the hypoxia-induced P-gp-mediated resistance to carfilzomib in MM cells. In vivo, in the MM tumor-initiation model, our study revealed that the HIF inhibitor, PX-478, significantly delayed the tumor progression and extended survival in which all control mice died between 42-52 days, while the experiment was stopped at 70 days, with all PX-478-treated mice still alive. In the established in vivo tumor model, low dose carfilzomib alone delayed the progression by BLI but did not improve survival (vehicle and carfilzomib-treated mice died between 21-28 days after treatment). Despite the fact that PX-478 did not decrease tumor progression as shown by BLI compared to the vehicle-treated mice, it significantly extended the survival of the mice (animals died between 38-48 days). The combination of carfilzomib and PX-478 significantly decreased the proliferation of tumor shown by BLI (less than 5% of the growth at day 28), as well as considerable increase in survival (the experiment was stopped at 70 days with 100% of the group alive). Conclusions: We identified a novel resistance mechanism to carfilzomib in MM, in which hypoxia induces P-gp-mediated resistance to carfilzomib. Inhibition of the hypoxic response in MM cells by the HIF inhibitor reduced hypoxia-induced P-gp-mediated resistance to carfilzomib in MM cells in vitro, and delayed tumor progression significantly improving survival and response to carfilzomib in MM-bearing mice in vivo. Disclosures Azab: Verastem: Research Funding; Selexys: Research Funding; Karyopharm: Research Funding; Cell Works: Research Funding; Targeted Therapeutics LLC: Other: Founder and owner.

scholarly journals Progression and Differentiation of Alveolar Rhabdomyosarcoma Is Regulated by PAX7 Transcription Factor—Significance of Tumor Subclones

Cells ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 1870
Author(s):  
Klaudia Skrzypek ◽  
Grażyna Adamek ◽  
Marta Kot ◽  
Bogna Badyra ◽  
Marcin Majka
Keyword(s):  
Transcription Factor ◽  
Cell Lines ◽  
Migration Activity ◽  
Id Proteins ◽  
Rh30 Cell ◽  
Str Profile ◽  
And Migration

Rhabdomyosarcoma (RMS), is the most frequent soft tissue tumor in children that originates from disturbances in differentiation process. Mechanisms leading to the development of RMS are still poorly understood. Therefore, by analysis of two RMS RH30 cell line subclones, one subclone PAX7 negative, while the second one PAX7 positive, and comparison with other RMS cell lines we aimed at identifying new mechanisms crucial for RMS progression. RH30 subclones were characterized by the same STR profile, but different morphology, rate of proliferation, migration activity and chemotactic abilities in vitro, as well as differences in tumor morphology and growth in vivo. Our analysis indicated a different level of expression of adhesion molecules (e.g., from VLA and ICAM families), myogenic microRNAs, such as miR-206 and transcription factors, such as MYOD, MYOG, SIX1, and ID. Silencing of PAX7 transcription factor with siRNA confirmed the crucial role of PAX7 transcription factor in proliferation, differentiation and migration of RMS cells. To conclude, our results suggest that tumor cell lines with the same STR profile can produce subclones that differ in many features and indicate crucial roles of PAX7 and ID proteins in the development of RMS.

Simvastatin Induces Death of Multiple Myeloma Cell Lines

2004 ◽  
Vol 52 (5) ◽  
pp. 335-344 ◽  
Author(s):  
Naomi Gronich ◽  
Liat Drucker ◽  
Hava Shapiro ◽  
Judith Radnay ◽  
Shai Yarkoni ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Cell Death ◽  
Cell Lines ◽  
Myeloma Cell ◽  
Cytoplasmic Calcium ◽  
Multiple Myeloma Cell ◽  
Cycle Arrest ◽  
Rpmi 8226

BackgroundAccumulating reports indicate that statins widely prescribed for hypercholesteromia have antineoplastic activity. We hypothesized that because statins inhibit farnesylation of Ras that is often mutated in multiple myeloma (MM), as well as the production of interleukin (IL)-6, a key cytokine in MM, they may have antiproliferative and/or proapoptotic effects in this malignancy.MethodsU266, RPMI 8226, and ARH77 were treated with simvastatin (0-30 μM) for 5 days. The following aspects were evaluated: viability (IC50), cell cycle, cell death, cytoplasmic calcium ion levels, supernatant IL-6 levels, and tyrosine kinase activity.ResultsExposure of all cell lines to simvastatin resulted in reduced viability with IC50s of 4.5 μM for ARH77, 8 μM for RPMI 8226, and 13 μM for U266. The decreased viability is attributed to cell-cycle arrest (U266, G1; RPMI 8226, G2M) and cell death. ARH77 underwent apoptosis, whereas U266 and RPMI 8226 displayed a more necrotic form of death. Cytoplasmic calcium levels decreased significantly in all treated cell lines. IL-6 secretion from U266 cells was abrogated on treatment with simvastatin, whereas total tyrosine phosphorylation was unaffected.ConclusionsSimvastatin displays significant antimyeloma activity in vitro. Further research is warranted for elucidation of the modulated molecular pathways and clinical relevance.

scholarly journals EXTH-64. SMALL GTPASES IN MENINGIOMAS: PROLIFERATION, MIGRATION, SURVIVAL, POTENTIAL TREATMENT AND INTERACTIONS

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii101-ii101
Author(s):  
Christoph Kesseler ◽  
Julian Kahr ◽  
Natalie Waldt ◽  
Nele Stroscher ◽  
Josephine Liebig ◽  
...  
Keyword(s):  
Overall Survival ◽  
Cell Lines ◽  
Small Gtpases ◽  
Potential Treatment ◽  
Rock Inhibitor ◽  
Meningioma Cell ◽  
And Migration ◽  
Proliferation And Migration

Abstract PURPOSE To evaluate the role of the small GTPases RhoA, Rac1 and Cdc42 in meningiomas as therapeutic targets and their interactions in meningiomas. EXPERIMENTAL DESIGN We analyzed expression of GTPases in human meningioma samples and meningioma cell lines of various WHO grades. Malignant IOMM-Lee meningioma cells were used to generate shRNA mediated knockdowns of GTPases RhoA, Rac1 or Cdc42 and to study knockdown effects on proliferation and migration, as well as analysis of cell morphology by confocal microscopy. The same tests were used to investigate effects of the two inhibitors Fasudil and EHT-1864 of malignant IOMM-Lee, KT21 and benign Ben-Men cells and the effects of these drugs on IOMM-Lee knockdown cells. The effects of GTPase knockdowns and Fasudil treatment were studied in terms of overall survival by intracranial xenografts of mice. Potential interactions of GTPases regarding NF2, mTOR and FAK-Paxillin were examined. RESULTS Small GTPases were upregulated in meningiomas of higher tumor grades. Reduced proliferation and migration could be achieved by GTPase knockdown in IOMM-Lee cells. Additionally, the ROCK-inhibitor Fasudil and Rac1-inhibitor EHT-1864 reduced proliferation in different meningioma cell lines and reduced proliferation and migration independent of GTPase knockdowns/status. Moreover, overall survival in vivo could also be increased by knockdowns of RhoA and Rac1 as well as Fasudil treatment. GTPase expression was affected dependent on the NF2 status but effects were not very distinct, indicating that NF2 is not strongly involved in GTPase regulation in meningiomas. In terms of mTOR and FAK-Paxillin signaling, each GTPase changes those pathways in a different manner. CONCLUSION Small GTPases are important effectors in meningioma proliferation and migration in vitro as well as survival in vivo and their inhibition should be considered as potential treatment option.

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