scholarly journals Super-Enhancer Driven Regulation of CKS1B in Multiple Myeloma: Implications in Mediating Response to BET Inhibitor and Celmod Agent Combination

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2658-2658
Author(s):  
Aarif Ahsan ◽  
Ann Polonskaia ◽  
Chih-Chao Hsu ◽  
Chad C Bjorklund ◽  
Maria Ortiz Estevez ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
High Risk ◽  
Cell Lines ◽  
Publicly Traded ◽  
Chip Sequencing ◽  
Chromosome 1Q ◽  
Cycle Arrest ◽  
Super Enhancer ◽  
Sequencing Studies

Abstract Introduction: The Myeloma Genome Project (MGP) characterized the genomic landscape of patients with newly diagnosed multiple myeloma (NDMM) (Walker BA, et al. Blood 2018; 132[6]:587-597). Using a multi-omics unsupervised clustering approach, 12 molecularly-defined disease segments were identified (Ortiz M, et al. Blood 2018; 132[suppl 1]:3165). Here, we performed experimental validation of CDC28 Protein Kinase Regulatory Subunit 1B (CSK1B) that was identified as a putative target from the disease segment with poorest clinical outcome. CKS1B was selected for in-depth validation due to their role in cell cycle pathways associated with high-risk disease, biological mechanisms of chromosome 1q amplification and druggability. Methods: Association of CKS1B with outcomes was analyzed in NDMM patients, across relapses and with clinical outcome datasets from MGP and Mayo clinic. Inducible shRNAs of CKS1B and bromodomain containing protein 4 (BRD4, a member of the BET [bromodomain and extra terminal domain] family) were generated in MM cell lines. BRD4 and Aiolos ChIP-seq datasets were analyzed for binding on CKS1B gene. BRD4 inhibitors JQ1 and CC-90010 were utilized for inhibition studies in MM cell lines. Results: Higher expression of CKS1B was associated with significantly poorer PFS, OS, disease severity and relapse. Knock-down of CKS1B in MM cells led to a significant decrease in proliferation (P<0.001) and enhanced apoptosis in MM cell lines. BRD4-ChIP sequencing studies revealed that the expression of CKS1B was regulated by super-enhancer (SE) associated elements. As expected, two BRD4 inhibitors, JQ1 and CC-90010 and inducible BRD4 shRNAs downregulated the expression of CKS1B resulting in decreased proliferation, cell cycle arrest and apoptosis in MM cell lines. Furthermore, MM cell lines harboring chromosome 1q gain/amp showed higher sensitivity to BRD4 inhibition compared to cell lines with normal 1q copy number. Mechanistic studies revealed that BRD4inh and BRD4 shRNAs downregulated the expression of Aiolos and Ikaros in MM cell lines. Interestingly, Aiolos ChIP-sequencing studies demonstrated the binding of Aiolos at the transcriptional start sites of CKS1B with the transcriptional activation mark. The immunomodulatory agent (IMiD ®) pomalidomide (Pom) transcriptionally downregulated CKS1B in Pom-sensitive cells downstream of Aiolos, Ikaros degradation. Based on these mechanisms, IMiD agents, lenalidomide, Pom and the novel Cereblon E3 ligase modulating degrader (CELMoD ®) agent CC-92480 in combination with BRD4inh promoted a synergistic decrease in proliferation, cell cycle arrest and increase in apoptosis in both Pom-sensitive and -resistant cell lines. The combination of IMiD or novel CELMoD agent with BRD4inh also promoted deeper downregulation of CKS1B, Aiolos, Ikaros, c-Myc and survivin proteins with enhanced levels of apoptotic marker cleaved Caspase 3 as compared to single agents alone. Conclusions: In summary, we have identified CKS1B as a key target associated with poor outcome in MM patients. Translational studies suggest a profound downregulation of CKS1B and key pro-survival effector proteins following combination treatment with BRD4inh and IMiD agents/novel CELMoD agents resulting in synergistic anti-tumor effects. These data provide rationale for testing these agents in the clinic for high-risk and IMiD-relapsed patients. Figure: Changes in cell proliferation and protein levels of key signaling mediators were studied in K12PE cell line treated with increasing doses of Lenalidomide, Pomalidomide and CC-92480 in combination with JQ1. Figure 1 Figure 1. Disclosures Ahsan: BMS: Current Employment, Current equity holder in publicly-traded company. Polonskaia: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Hsu: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Bjorklund: BMS: Current Employment, Current equity holder in publicly-traded company. Ortiz Estevez: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Towfic: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Bahlis: Takeda: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; GlaxoSmithKline: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Genentech: Consultancy; Pfizer: Consultancy, Honoraria; BMS/Celgene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria. Pourdehnad: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties: No royalty. Flynt: BMS: Current Employment, Current equity holder in publicly-traded company. Ahsan: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Thakurta: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties.

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 Induction of Apoptosis, Autophagy and Ferroptosis by Thymus vulgaris and Arctium lappa Extract in Leukemia and Multiple Myeloma Cell Lines

Molecules ◽  
2020 ◽  
Vol 25 (21) ◽  
pp. 5016
Author(s):  
Aveen N. Adham ◽  
Mohamed Elamir F. Hegazy ◽  
Alaadin M. Naqishbandi ◽  
Thomas Efferth
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Cell Death ◽  
Ethyl Acetate ◽  
Cell Cycle Arrest ◽  
Cell Lines ◽  
Thymus Vulgaris ◽  
Arctium Lappa ◽  
Cycle Arrest ◽  
Induction Of Apoptosis

Thymus vulgaris and Arctium lappa have been used as a folk remedy in the Iraqi Kurdistan region to deal with different health problems. The aim of the current study is to investigate the cytotoxicity of T. vulgaris and A. lappa in leukemia and multiple myeloma (MM) cell lines and determine the mode of cell death triggered by the most potent cytotoxic fractions of both plants in MM. Resazurin assay was used to evaluate cytotoxic and ferroptosis activity, apoptosis, and modulation in the cell cycle phase were investigated via Annexin V-FITC/PI dual stain and cell-cycle arrest assays. Furthermore, we used western blotting assay for the determination of autophagy cell death. n-Hexane, chloroform, ethyl acetate, and butanol fractions of T. vulgaris and A. lappa exhibited cytotoxicity in CCRF-CEM and CEM/ADR 5000 cell lines at concentration range 0.001–100 μg/mL with potential activity revealed by chloroform and ethyl acetate fractions. NCI-H929 displayed pronounced sensitivity towards T. vulgaris (TCF) and A. lappa (ACF) chloroform fractions with IC50 values of 6.49 ± 1.48 and 21.9 ± 0.69 μg/mL, respectively. TCF induced apoptosis in NCI-H929 cells with a higher ratio (71%), compared to ACF (50%) at 4 × IC50. ACF demonstrated more potent autophagy activity than TCF. TCF and ACF induced cell cycle arrest and ferroptosis. Apigenin and nobiletin were identified in TCF, while nobiletin, ursolic acid, and lupeol were the main compounds identified in ACF. T. vulgaris and A. lappa could be considered as potential herbal drug candidates, which arrest cancer cell proliferation by induction of apoptosis, autophagic, and ferroptosis.

Aptamer TY04 Inhibits Multiple Myeloma Cell Growth Via Cell Cycle Arrest

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 5390-5390
Author(s):  
Jing Liu ◽  
Hong-Juan Dai ◽  
Bian-Ying Ma ◽  
Jian-Hui Song ◽  
Hui-yong Chen ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Cell Growth ◽  
Cell Lines ◽  
Myeloma Cell ◽  
Multiple Myeloma Cell ◽  
Cycle Arrest ◽  
M Phase ◽  
Target Molecules

Abstract Multiple myeloma (MM), also known as plasma cell myeloma, is characterized by accumulation of clonal plasma cells in the bone marrow and overproduction of monoclonal immunoglobulin (Ig) in the blood or urine. MM accounts for approximately 10% of all hematologic malignancies. Despite recent advances in the understanding and treatment of this disease, MM remains an incurable disease in the vast majority. With conventional chemotherapy, the 5-year median survival rate for MM patients is approximately 25%. Aptamers are single-stranded RNA or DNA sequences that bind to target molecules with high affinity and specificity. Compared with antibodies, aptamers have unique advantages including easy chemical synthesis and modification, low toxicity, lack of immunogenicity, and rapid tissue penetration, Based on these advantages, aptamers show great potential for therapeutic application. The aptamer TY04 is a single-stranded DNA (ssDNA) generated by a method named cell-based systematic evolution of ligands by exponential enrichment (cell-SELEX), We found TY04 strongly inhibited the growth of multiple myeloma cell lines including MM1.S, NCI-H929, KM3 and OPM2,The concentration of TY04 to inhibit 50% cell growth (IC50) on MM1.S was 3.89 μM. In contrast, TY04 had no effect on the growth of non-tumor cell lines — immortal B lymphoblastoid cell lines. Next, we used MM1.S cell line as the model to study the mechanism of TY04 anti- multiple myeloma. Flow cytometry analysis showed that TY04 with the sequence specifically bind to MM1.S cells when compared with unselected ssDNA library control. To investigate whether the target molecules of TY04 are membrane proteins on cell surface, MM1.S cells were treated with trypsin and proteinase k for 2 or 10 minutes before incubation with TY04. The result revealed that TY04 lost partly recognition ability on treated cells, indicating that the target molecules were most likely membrane proteins. Furthermore, we evaluated the cell cycle distribution of MM1.S after TY04 treatment. We found that TY04 significantly caused cell-cycle arrest in G2/M phase. The percentage of G2/M phase cells increased from 30.1±1.56 to 53.2±6.36. To identify the underlying molecular mechanism, G2/M-related proteins were assayed by flow cytometry. Following TY04 treatment, a concomitant inhibition of ERK1/2, cyclin B, CDK1 and γ-tubulin expression occurred. Meanwhile, human cell cycle PCR array was used to analyze the expression of 84 genes key to cell cycle regulation in TY04-treated MM1.S cells. Our results indicated that aptamer TY04 decreased the genes expression of CCNB1, CCNB2, BIRC5, BRCA1 and CCNH, which were involved in the progress of G2/M phase. All these results are significant in that they provide a framework for further exploring the use of TY04 as a novel anti-multiple myeloma agent. Disclosures: No relevant conflicts of interest to declare.

Effect of the specific P53 stabilizer CBS9106 on multiple myeloma (MM)

2009 ◽  
Vol 27 (15_suppl) ◽  
pp. 8601-8601
Author(s):  
H. Ikeda ◽  
T. Hideshima ◽  
G. Perrone ◽  
Y. Okawa ◽  
N. Raje ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Protein Expression ◽  
Tumor Cells ◽  
Cell Lines ◽  
Primary Tumor ◽  
P53 Protein ◽  
Wild Type ◽  
Cycle Arrest ◽  
P53 Protein Expression

8601 Background: The mutations of P53 tumor suppressor protein are associated with progressive in Multiple Myeloma (MM), conversely, stabilization of P53 leads to cell cycle arrest and apoptosis. In this study, we examined p53 protein expression and demonstrated the effect of P53 stabilization using a novel specific P53 stabilizer CBS9106 in MM. Method: We examined P53 protein expression using Immunoblot analysis, as well as the growth inhibitory effect of CBS9106 in MM cell lines and primary tumor cells from MM patients. We also defined whether CBS9106 can overcome the growth promoting effect of exogenous cytokines and bone marrow stroma cells (BMSCs) using [3H]-thymidine uptake assay. Results: Expression of P53 protein was observed in 3/3 primary tumor cells from MM patients and 6/6 MM cell lines. CBS9106 at low nM levels triggered cytotoxicity against p53 wild type MM cell lines and primary tumor cells from MM patients, associated with phosphorylation of P53 (serine15 and 20). In contrast, CBS9106 did not affect the survival of normal peripheral blood mononuclear cells from healthy volunteers at concentrations as high as 10 μM. This agent also induced G1 cell cycle arrest, followed by apoptosis associated with cleavage of caspase-3, -8, -9 and PARP. Neither growth stimulating cytokines (IL-6 and IGF-1) nor BMSCs protected against apoptotic effect of CBS9106. Moreover, we demonstrate that combination of CBS9106 with MDM2 inhibitor Nutrin3 or proteasome inhibitor bortezomib induces synergistic anti-MM activity in both P53 wild type MM cell lines and primary tumor cells from MM patients. Conclusions: Stabilizing P53 by CBS9106 represents a novel promising p53-based therapy in MM. These results provide the preclinical framework supporting evaluation of CBS9106 in clinical trials to improve patient outcome in MM. No significant financial relationships to disclose.

Valproic Acid Inihibts Histone Deacetylases and Proliferation and Induces Cell Cycle Arrest and Apoptosis in Multiple Myeloma.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5165-5165
Author(s):  
Martin Kaiser ◽  
Ulrike Heider ◽  
Ivana Zavrski ◽  
Jan Sterz ◽  
Kurt Possinger ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Valproic Acid ◽  
Cell Lines ◽  
Myeloma Cell ◽  
G1 Phase ◽  
Dependent Manner ◽  
Myeloma Cells ◽  
Cycle Arrest ◽  
Dose Dependent

Abstract Multiple myeloma remains an incurable disease in the majority of the patients and novel treatment strategies are urgently needed. A new class of drugs, the histone deacetylase (HDAC) inhibitors take influence in epigenetic modifications and have antiproliferative effects in some malignancies. Valproic acid (VPA) is an anticonvulsant drug and was recently shown to inhibit HDACs and suppress tumor growth. The drug is currently being evaluated in clinical studies in acute myeloid leukemia. Its effects on myeloma cells are unknown. The aim of this study was to evaluate the effects of VPA on proliferation, apoptosis and HDAC inhibition in multiple myeloma cell lines as well as in sorted human bone marrow multiple myeloma cells. Myeloma cell lines, OPM-2, NCI-H929, LP-1, and freshly isolated multiple myeloma cells from bone marrow aspirates were exposed to different concentrations of VPA for 4 to 72 hours. Cell proliferation, cell cycle distribution and apoptosis were assayed in reaction to the treatment. Proliferation decreased noticeably and apoptosis was induced in a dose-dependent manner in multiple myeloma cell lines as well as in freshly sorted primary myeloma cells. After 48 hours of incubation with VPA at 1 mM, approximately 46%, 52% and 25% of OPM-2, NCI-H929 and LP-1 cell lines had undergone specific apoptosis, respectively. Freshly sorted primary bone marrow myeloma cells from patients showed also specific apoptosis. In cell cycle analysis by flow cytometry, the population of cells in the G0/G1 phase increased, whereas cells in the S phase decreased in a time and dose dependent manner. Incubation of the cell line OPM-2, for example, with 1 mM VPA for 48 hours decreased the proportion of cells in the S phase from 39 % to 6 % of the total cell count and increased cells in the G0/G1 phase from 49 % to 85 %. Acetylation of histones and expression of cyclin D1 and the cell cycle regulators p21 and p27 were studied by western blot. Histone acetylation and p21 concentrations increased after VPA treatment whereas levels of p27 remained constant. A decrease in cyclin D1 concentrations was observed. Subapoptotic doses of VPA significantly decreased the production of VEGF in OPM-2 cell line. These data show that treatment with valproic acid effectively inhibits histone deacetylase activity, leading to the accumulation of acetylated histones in multiple myeloma cells. Parallel upregulation of cell cycle inhibitors like p21WAF1 was observed, together with a reduction of cyclin D1 levels. Myeloma cell proliferation was inhibited in a time and dose dependent manner and cell cycle arrest in the G0/G1 phase was induced by VPA treatment. VPA potently induced apoptosis in all human myeloma cell lines as well as in sorted primary multiple myeloma cells in a dose and time dependent manner. These results show for the first time that VPA acts as an HDAC inhibitor in multiple myeloma cells, induces G1 cell cycle arrest, potently inhibits tumor growth and markedly induces apoptosis. In addition to its direct antitumor effect, valproic acid may exert an antiangiogenic effect by reducing VEGF production in myeloma cells. These data provide the framework for clinical studies with valproic acid in multiple myeloma.

β1 Integrin Adhesion Enhances IL-6 Mediated STAT3 Signaling in Multiple Myeloma Cells: Implications for Microenvironment Influence on Tumor Cell Survival and Proliferation.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1706-1706
Author(s):  
Kenneth H Shain ◽  
Danielle Yarde ◽  
Mark Mead ◽  
Lori Hazlehurst ◽  
William S Dalton
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Cell Cycle Arrest ◽  
Cell Lines ◽  
Genetic Alterations ◽  
Β1 Integrin ◽  
Cycle Arrest ◽  
Stat3 Signaling ◽  
Stat3 Phosphorylation ◽  
In Cells

Abstract Multiple Myeloma (MM) is a B cell malignancy characterized by the monoclonal expansion of plasma cells. Although numerous genetic alterations have been implicated in MM pathogenesis, it is widely hypothesized that the bone marrow (BM) microenvironment contributes to MM cell pathogenesis. The BM microenvironmental components, interleukin (IL)-6 and fibronectin (FN), have individually been shown to influence the proliferation and survival of MM cells; however, in vivo these effectors most likely work together. We examined signaling events, cell cycle progression, and levels of drug response in MM cells either adhered to FN via β1 integrins, stimulated with IL-6, or with the two combined. IL-6 and FN adhesion have been demonstrated to protect cells from a host of cytotoxic stimuli suggesting co-stimulation of MM cell lines with IL-6 and FN-adhesion may confer a greater protection against chemotherapeutics than either effector alone. However, MTT cytotoxicity assays demonstrate that although adhesion to FN provides significant protection against treatment with mitoxantrone or doxorubicin (p=0.0002 and p=<0.0001 respectively), the addition of IL-6 provides no further protection. These findings were corroborated by analysis of drug-mediated apoptosis using FCM by Annexin-V/7-AAD. In regards to cell cycle kinetics, our laboratory has previously demonstrated that adhesion of the 8226 MM cell line to FN mediated a p27Kip1 dependent G0/G1 cell cycle arrest. As predicted, BrdU/PI analysis of 8226 cells adhered to FN for 24 hours results in an increased number of cells in G0/G1 relative to cells maintained in suspension (p=0.0028). In contrast, when cells were adhered to FN in the presence of IL-6 no accumulation of cells in G0/G1 was observed, with levels similar to that observed in cells maintained in suspension with or without stimulation by IL-6. Our studies demonstrated that the G1/S cell cycle arrest associated with FN adhesion of MM cell lines was overcome when IL-6 was added; however, the cell adhesion mediated drug resistance (CAM-DR) was maintained in the presence of IL-6. Investigation of the biochemical signaling following concomitant exposure of MM cells to IL-6 and FN adhesion revealed a synergistic increase in STAT3 phosphorylation, nuclear translocation and DNA-binding as compared to either IL-6 or FN-adhesion alone in four MM cell lines. STAT3 phosphorylation was increased in cells adhered to FN in an IL-6 dose dependent manner. Electrophoretic mobility shift assay demonstrated a parallel 3-fold increase in STAT3/DNA complexes in cells adhered to FN relative to cells in suspension. To further characterize the receptor proximal affects of FN adhesion on IL-6 signaling we immunoprecipitated the IL-6R complex with antisera to gp130. Immunoprecipitation of gp130 revealed enhanced tyrosine phosphorylation of the gp130/Jak family complexes following stimulation FN-adhered RPMI 8226 MM cells with IL-6. Consistent with increased phosphorylation of the receptor complex, increased levels of phospho-STAT3 were identified associated with gp130 under co-stimulatory conditions relative to IL-6 or FN adhesion alone. Interestingly, immunoprecipitation with gp130 antibodies also revealed an association between STAT3 (non-phosphorylated) and gp130 in the absence of IL-6 stimulation in cells adhered to FN. These results suggest that adhesion to FN facilitates an IL-6-independent association between gp130 and STAT3, resulting in enhanced STAT3 signaling. Taken together, these data demonstrate a novel mechanism by which collaborative signaling by β1 integrin and gp130 confer an increased survival advantage to MM cells.

Hypomethylating Agents Correct Subcellular Localization of FOXO3a and (re)Establish Expression of Its Transcriptional Targets In AML and MDS- Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2173-2173
Author(s):  
Sylvain Thepot ◽  
Elodie Lainey ◽  
Marie Sebert ◽  
Lionel Ades ◽  
Claude Gardin ◽  
...  
Keyword(s):  
Cell Cycle ◽  
High Risk ◽  
Subcellular Localization ◽  
Cell Cycle Arrest ◽  
Cell Lines ◽  
Nuclear Translocation ◽  
Hypomethylating Agents ◽  
Transcriptional Targets ◽  
Cycle Arrest ◽  
Functional Relevance

Abstract Abstract 2173 Background: We previously found deregulation of DNA-damage response (DDR) signaling in MDS and AML, notably the “uncoupling” between upstream proteins (ATM) and downstream effectors (Chk-1/2) in high-risk MDS and AML (Boehrer et al., Oncogene 2009). Here, we assessed if and to which degree the hypomethylating agents azacitidine (AZA) and decitabine (DAC), widely used agents in the treatment of high-risk MDS and AML, can overcome these defects. Methods: MDS (MOLM-13)- and AML- (HL-60, KG-1)-derived cell lines were incubated with AZA (1-2μM) and DAC (1-2μM). The drugs capacity to induce apoptosis (DiOC6(3)/PI), cell cycle arrest (PI) and their impact on the expression level (immunoblot) and subcellular localization (immunoflourescence) of DDR-related proteins were concomitantly assessed. Functional relevance was determined by co-incubation with biochemical inhibitors of ATM (KU-55933), ATM/ATR (caffeine), Chk-1 (UCN-01), and Chk-2 (NSC-109555). Most important findings were recapitulated on MDS- and AML- patient cells (n=5) after selection for CD34-positvity by immunomagnetic beads. Results: As previously described, both drugs induced dose- and time-dependent apoptosis and cell cycle arrest in G2/M. Noteworthy, whereas both drugs completely abrogated expression of DNA methyltransferases 1 and 3a as early as 4h of incubation with 2μM (abrogation sustained at 24 and 48h), they differentially affected DDR-related signaling. Indeed, the increased capacity of DAC (as compared to AZA) to induce G2/M-arrest (G2/M-increase at 48h DEC: 20%, AZA: +6%) was accompanied by an earlier and more efficient activation of the checkpoint-kinases-1 and -2 (phosphorylation of Chk-1-Ser317 and Chk-2-Ser68) as well as their downstream target y-H2AX (phosphorylation on Ser139). Noteworthy, DAC (and to a lesser extent AZA) modified activation of FOXO3a (diminished phosphorylation on Ser253) and concomitantly enabled nuclear translocation of non-phosphorylated FOXO3a. In addition, nuclear translocation of FOXO3a was accompanied by upregulation of its transcriptional targets known to confer G2/M- arrest (p21, p27) and apoptosis (BH3-only protein BIM). Confirming the functional relevance of our findings, we found that inhibition of ATM (KU-55933) or ATM/ATR (caffeine) had little to no impact on AZA /DAC-induced apoptosis or inhibition of cell cycle progression, whereas inhibition of Chk-1 by UCN-01 (and to a lesser degree inhibition of Chk-2 by NSC-1095555) abrogated G2/M-arrest induced by hypomethylating agents. Finally we showed that both DAC and AZA (2μM) can induce the cytoplasmic-nuclear shift of FOXO3a in MDS- and AML-derived cell lines and to a lesser extent in patient blasts, an effect observed as early as 4h of the incubation period. Conclusions: Hypomethylating agents can correct aberrant DDR-signaling in AML-and MDS blasts, and can abrogate constitutive phosphorylation of FOXO3a thereby “correcting” its aberrant cytoplasmic localization and (re-)establishing its function as a transcriptional regulator. This possible mode of action of hypomethylating agents may be important considering that aberrant phosphorylation and localization of FOXO3a is an adverse prognostic factor in AML, and that correction of its deregulation has been described as an attractive therapeutic aim. Disclosures: Fenaux: CELGENE, JANSSEN CILAG, AMGEN, ROCHE, GSK, NOVARTIS, MERCK, CEPHALON: Consultancy.

scholarly journals Diallyl thiosulfinate enhanced the anti-cancer activity of dexamethasone in the side population cells of multiple myeloma by promoting miR-127-3p and deactivating the PI3K/AKT signaling pathway

BMC Cancer ◽  
2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Wenfeng He ◽  
Yonghui Fu ◽  
Yongliang Zheng ◽  
Xiaoping Wang ◽  
Bin Liu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Cell Proliferation ◽  
Cell Cycle Arrest ◽  
Signaling Pathway ◽  
Cell Lines ◽  
Colony Formation ◽  
Side Population ◽  
Mtor Signaling Pathway ◽  
Cycle Arrest

Abstract Background Side population (SP) cells, which have similar features to those of cancer stem cells, show resistance to dexamethasone (Dex) treatment. Thus, new drugs that can be used in combination with Dex to reduce the population of SP cells in multiple myeloma (MM) are required. Diallyl thiosulfinate (DATS, allicin), a natural organosulfur compound derived from garlic, has been shown to inhibit the proliferation of SP cells in MM cell lines. Therefore, we investigated the effect of a combination of DATS and Dex (DAT + Dex) on MM SP cells. Methods SP cells were sorted from MM RPMI-8226 and NCI-H929 cell lines using Hoechst 33342-labeled fluorescence-activated cell sorting. The growth of SP cells was evaluated using the cell counting kit-8 assay. Cell cycle and apoptosis assays were conducted using a BD Calibur flow cytometer. miRNA expression was measured using quantitative reverse transcription-polymerase chain reaction. Phosphoinositide 3-kinase (PI3K), phosphorylated AKT (p-AKT), AKT, p-mechanistic target of rapamycin (mTOR), and mTOR levels were measured using western blot analysis. Results Our results showed that the combination of DATS+Dex inhibited sphere formation, colony formation, and proliferation of MM SP cells by inducing apoptosis and cell cycle arrest in the G1/S phase. In addition, the combination of DATS+Dex promoted miR-127-3p expression and inhibited PI3K, p-AKT, and p-mTOR expression in SP cells. Knockdown of miR-127-3p expression weakened the effect of DATS+Dex on cell proliferation, colony formation, apoptosis, and cell cycle of MM SP cells. Additionally, knockdown of miR-127-3p activated the PI3K/AKT/mTOR signaling pathway in MM SP cells cotreated with DATS+Dex. Conclusion We demonstrated that cotreatment with DATS+Dex reduced cell proliferation, promoted apoptosis, and caused cell cycle arrest of MM SP cells by promoting miR-127-3p expression and deactivating the PI3K/AKT/mTOR signaling pathway.

The Novel Histone Deacetylase Inhibitor RAS2410 (Resminostat) Interferes with Important Signalling Pathways and Induces Apoptosis in Multiple Myeloma Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4928-4928
Author(s):  
Philipp Baumann ◽  
Sonja Mandl-Weber ◽  
Felix Meinel ◽  
Ruediger Jankowsky ◽  
Fuat S. Oduncu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Cell Cycle Arrest ◽  
Histone Deacetylase ◽  
Cell Lines ◽  
Synergistic Effects ◽  
In Vivo Studies ◽  
The Novel ◽  
Cycle Arrest

Abstract Abstract 4928 Inhibition of histone deacetylase (HDAC) is a promising target for novel, anti-myeloma agents. In this study we investigated the biologic effects of the novel HDAC inhibitor RAS2410 (also known as “4SC-201”, “resminostat”) on Multiple Myeloma (MM) cells in vitro. RAS2410 is a potent, direct inhibitor of HDACs 1, 3 and 6 (IC50 = 43-72nM) representing the HDAC classes I and II. Accordingly, RAS2410 induces hyperacetylation of histone H4 in MM cells. Low micromolar concentrations of RAS2410 abrogate cell growth and strongly induce apoptosis (IC50 = 2.5-3μM in 3 out of 4 cell lines) in MM cell lines (NCI-H929, U-266, RPMI-8226, OPM-2) as well as in primary MM cells isolated from patients. At 1μM, RAS2410 induces G0/G1 cell cycle arrest in 3 out of 4 MM cell lines associated with decreased levels of cyclin D1, cdc25a, Cdk4, pRb and p53 as well as upregulation of p21. This cell cycle arrest is reflected by an inhibition of cell proliferation. RAS2410 decreases phosphorylation of 4EBP-1 and P70S6K indicating that RAS2410 induces apoptosis by interfering with Akt pathway signalling downstream of Akt. Treatment with RAS2410 results in increased protein levels of Bim and Bax and decreased levels of Bcl-xL. Caspases 3, 8 and 9 are activated by RAS2410. Furthermore, additive and synergistic effects in terms of apoptosis induction are observed for combinations of RAS2410 with melphalan, doxorubicin and the proteasome inhibitors bortezomib and S2209. In conclusion, we have identified potent anti-myeloma activity for the novel HDACi RAS2410. This study has yielded further insight into the biological sequelae of HDAC inhibition in MM and provides the rationale for in vivo studies and clinical trials using RAS2410 to improve patient outcome in MM. Disclosures Jankowsky: 4SC: Employment. Schmidmaier:4SC : Research Funding.

A Novel Class of Sulfonanilides Entering Clinical Trials for Targeted Treatment of Multiple Myeloma: Dual-Mechanism Compounds Inhibiting HIF1A-Signaling and Inducing Apoptosis

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2987-2987 ◽  
Author(s):  
Dirk Hose ◽  
Anja Seckinger ◽  
Hartmut Goldschmidt ◽  
Tobias Meißner ◽  
Blanka Leber ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Cycle Arrest ◽  
Cell Lines ◽  
Mechanism Of Action ◽  
Myeloma Cell ◽  
Cycle Arrest ◽  
Dual Mechanism

Abstract Abstract 2987 Background. We have recently shown HIF1A to be expressed in 95.4% of CD138-purified myeloma cell samples from previously untreated patients (n= 329), with significantly higher [lower] expression in case of presence of t(4,14) [hyperdiploidy] vs. patients without the respective aberration. This makes HIF1A an interesting target in myeloma treatment. Additionally, we have shown about 40% of myeloma cell samples to have a proliferation-index above the median plus three standard-deviations of normal bone-marrow plasma cells, and we and others have proven proliferation to be associated with adverse prognosis in myeloma. Here, we report on 2 members of a novel class of sulfonanilides, their preclinical activity and pharmacology, and their dual mechanism of action, targeting HIF1A-signaling and inducing apoptosis via cell cycle arrest and tubulin depolymerization. Patients and Methods. The effect of the novel sulfonanilides ELR510444 and ELR510552 on the proliferation of 20 human myeloma cell lines and the survival of 5 primary myeloma cell-samples cultured within their microenvironment were tested. The results of efficacy studies in in two murine models (RPMI8226-xenograft-model and 5T33-model) are also presented. The mechanism of action was investigated using a variety of in-vitro assays (see below). Results. Preclinical activity in Myeloma. i) The sulfonanilides ELR510444 and ELR510552 completely inhibit proliferation of 20/20 tested myeloma cell lines at low nM concentrations and ii) induce apoptosis in 5/5 primary myeloma cell-samples at 6.4 – 32 nM concentration, without major effect on the bone marrow microenvironment. iii) They significantly inhibit tumor growth (xenograft; RPMI8226 mouse model, 6 mg p.o. bid for ELR510444, 15 mg p.o. bid for ELR510552) and bone marrow infiltration (5T33-model; ELR510444, 6 mg/kg p.o. bid × 4d, rest 3d (cycle)). Mechanism of action. Apoptosis induction and G2/M-block. i) Both compounds lead to caspase-3/7 activation and subsequent apoptosis with cellular EC50 values of 50–100 nM. ii) The compounds induce an initial cellular arrest in G2/M and a significant tubulin depolymerizing effect, followed by an increase in a sub-G1 (apoptotic) population after 24h. HIF1A-inhibition. i) Both compounds show a potent inhibition of HIF1A signaling in a cell based reporter assay (HRE-bla HCT-116) at EC50s of 1–25nM, whereas ii) at concentrations of 1 μ M, neither of the compounds shows an effect in assay systems monitoring the JAK/STAT, NFκB, PI3K/AKT/FOXO or Wnt/β-catenin-signaling pathways. iii) Kinase inhibition profiling showed no significant inhibition at 1μ M in two assays assessing 100 (Invitrogen) and 442 (Ambit) kinases, respectively. Pre-clinical pharmacology. Single dose exposure of 25 mg p.o. yields a maximum concentration of 1.1 μ M with a half life time of 3.6 hours (ELR510444) and 2.7 μ M and 6.6 h (ELR510552) in mice, respectively. The compounds are well-tolerated at levels that are significantly above the in vitro EC50 in all myeloma cell lines and primary samples tested. Conclusion. ELR510444 and ELR510552 are very active on all tested myeloma cell lines and primary myeloma cells without major impact on the bone marrow microenvironment, and show activity in two different mouse models. The compounds inhibit HIF1A-signaling and induce apoptosis via cell cycle arrest and tubulin depolymerization. Preclinical pharmacology data show favorable in vivo profiles with exposure levels in mice significantly higher than concentrations required for in vitro activity. Therefore, this novel class of compounds represents a promising weapon in the therapeutic arsenal against multiple myeloma entering a phase I/II trial within the next year. Disclosures: Leber: ELARA Pharmaceuticals GmbH: Employment. Janssen:ELARA Pharmaceuticals GmbH: Employment. Lewis:ELARA Pharmaceuticals GmbH: Employment. Schultes:ELARA Pharmaceuticals GmbH: Employment.

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