Identification of Gene Networks Associated with the Anti-Leukemic Effect of Anti-Inflammatory Drugs on Acute Myeloid Leukemia Cell Lines

Abstract Introduction Despite recent advances in therapy, acute myeloid leukemia (AML) remain a medical challenge with high morbidity and mortality rates. For most patients, allogeneic hematopoietic stem cell transplantation remain the only curative option, but due to the advanced age at diagnosis, a significant proportion of patients are not elegible to this form of therapy. Nevertheless, novel therapies are warranted. There is preclinical evidence that anti-inflammatory compounds, such as COX-2 inhibitors and steroids, may have anti-neoplastic activity in different tumor types, including AML; nevertheles the mechanisms associated with its anti-neoplastic activity are not clear. Therefore, the aim of this work was to evaluate the anti-leukemic effect of the anti-inflammatory compounds nimesulide and prednisolone in AML cell lines and to identify genes and molecular pathways associated with cytotoxicity through transcriptome analysis. Methods The leukemic cell lines HL-60, THP-1, OCI-AML2 and OCI-AML3 were treated with nimesulide and prednisolone at 100 µM alone and in combination and with cytarabine at 2.5 µM. Twenty four hours after treatment , we measured the amount of cell death using Annexin V Apoptosis Detection Kit FITC (ThermoFisher) and the cell cycle was analized after fixing the cells with 70% alcohol and incubation with propidium iodide (1mg/ml) and RNAse (10mg/ml). In another experiment, we harvested the cells after 4 hours of treatment for transcriptome analysis. RNA was extracted from control (DMSO) and treatment groups (1 - nimesulide, 2 - prednisolone , 3 - nimesulide and prednisolone) with RNeasy Mini Kit (Qiagen). The Illumina® NEBNext® Ultra II Directional RNA Library Prep Kit was used for library preparation, following the manufacturer protocol using Poly(A) mRNA Magnetic Isolation Module. Equimolar amount of libraries was sequenced using an Illumina NextSeq 500, following the manufacturer's instructions, on the Oklahoma Medical Research Foundation Genomics Core (USA). The sequences obtained with the RNA-Seq technique were aligned in the human genome of reference GRCh37.75 by the software Spliced Transcripts Alignment to a Reference (STAR) v2.5 and to obtain normalized counts in FPKM, the software Expectation-Maximization (RSEM) v1.3.0 was used. To identify network of genes correlated with the treatment (modules), we used the Weighted Correlation Network Analysis (WGCNA). Functional enrichment analysis of the WGCNA differentially expressed modules was performed using the Integrated Annotation, Visualization and Discovery Database (DAVID) v6.8 in order to correlate with biological processes. Results In the cell cycle analysis, we observed a significant increase (p < 0.05) in the sub-G0 phase (cell death) after treatment with nimesulide alone, and in combination with prednisolone (figure 1). No effect was observed in the prednisolone only group. The cell cycle effect induced by nimesulide on HL-60 and OCI-AML2 was similar to the induced by cytarabine, a standard chemotherapy agent for AML that in known to induce arrest in the S phase. In addition, the cell line arrest in THP-1 was greater with nimesulide than with cytarabine, while OCI-AML3 was less sensitive to both nimesulide and cytarabine. Regarding cell death mechanism, treatment with nimesulide induced predominantly an increase in late apoptosis that was potentiated after combined treatment with nimesulide and cytarabine (figure 2). After the demonstration of cell cycle arrest and apoptosis induction after treatment with nimesulide, we performed whole transcriptome sequencing followed by WGCNA analysis. We have identified gene modules that were significantly correlated with anti-inflammatory treatments, being 1 module down-regulated (lightyellow with p = 0.00052) and 2 modules up-regulated (lightcyan with p = 0 .00025 and tan with p = 0.000038). Analysis of functional enrichment using DAVID showed up-regulation of gene networks associated with apoptotic processes and autophagy and down- regulation of gene networks associated with cell cycle and RNA splicing pathways Conclusions The COX-2 inhibitor nimesulide caused cell cycle arrest and apoptosis in AML cell lines and potentiated the cytotoxic effects of cytarabine. This treatment was associated with up- regulation of autophagy and apoptosis and down-regulation of cell cycle and RNA splicing gene networks. Figure 1 Figure 1. Disclosures Santos: Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees. Campregher: Astellas: Consultancy.

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Functional Defects In Neutrophils Derived From Ezh2 Null Mice

Blood ◽

10.1182/blood.v122.21.1556.1556 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1556-1556

Author(s):

Albert Perez-Ladaga ◽

Bennett Caughey ◽

Huafeng Xie ◽

Stuart H. Orkin ◽

David B. Sykes ◽

...

Keyword(s):

Cell Cycle ◽

Cell Death ◽

Progenitor Cells ◽

Board Of Directors ◽

Cell Lines ◽

Resting Cells ◽

Surface Markers ◽

Advisory Committees ◽

Spontaneous Cell Death

Abstract Introduction We investigate the role of Ezh2 in neutrophil function using murine progenitor cells differentiated into neutrophils lacking the Ezh2 gene. Ezh2 is the catalytic component of the polycomb repressive complex 2, which methylates lysine 27 of histone H3. It is frequently disrupted in myelodysplastic syndromes (MDS) leading to loss of function (Ernst et al., 2010). Mutations in EZH2 are found in 6% of MDS patients and while not strongly linked to cytopenias or blast proportion, they are independently associated with worse overall survival compared to patients with wildtype EZH2 (Bejar R. et al., 2011 and 2012). We hypothesize that Ezh2 mutations may cause qualitative defects in myeloid cells that impact their function and could contribute to the adverse prognosis observed in EZH2 mutant MDS. Methods Bone marrow from Ezh2 null (Ezh2-/-) and littermate control mice (WT) were transduced with HOXB8 fused to the estrogen receptor ligand-binding domain to produce immortalized myeloid progenitor cells. Removal of estrogen from the media allows these cells differentiate into mature neutrophils (Wang G.G., 2006). Differentiated cells were characterized for surface markers by flow cytometry and for gene expression by PCR of mRNA. Spontaneous cell death was measured by annexin/PI staining. Cell cycle patterns were determined by measuring the red emission of PI. Chemotactic function was assessed by counting cells that migrated across a transwell in presence/absence of the attractant zymosan. For phagocytosis experiments, cells were incubated with Fluoresbrite YG carboxylate beads at 37°C or 4°C. Reactive oxygen species (ROS) generation was measured by the oxidation of dihydrorhodamine 123 into fluorescent rhodamine 123. Results Estrogen withdrawal caused differentiation of both WT and Ezh2-/- lines into cells with mature neutrophil morphology after six days (Figure 1a). Both differentiated lines expressed the neutrophil surface markers CD11b and CD62L and the neutrophil-specific genes lactoferrin and Itgb2l. Ezh2 -/- cells had an increased rate of spontaneous cell death compared to WT in undifferentiated (32.81% vs. 20.33%) and mature cells (32.82% vs. 14.23%). Nevertheless, both progenitor cell lines showed similar cell cycle patterns, demonstrating that Ezh2 absence had no other effect on cell cycle progression. Ezh2 -/- neutrophils failed to migrate towards zymosan (Figure 1b). Expression of Tlr2, which binds zymosan, and other Toll-like receptors (Tlr4/5/9) were similar between the differentiated cell lines. Cells incubated with FITC-zymosan at 37°C showed no fluorescence differences between cell lines, indicating similar adherence. Experiments with neutrophils from an MDS patient with homozygous EZH2 mutations demonstrated a similar migration defect. Additional studies in MDS patient samples are ongoing and will be presented. Phagocytosis was reduced in Ezh2-/-cells. Unstimulated, the number of cells ingesting and adhering YG-beads was significantly greater with WT cells than with Ezh2-/-cells. When activated with fMLP, both lines showed increased adherence of YG-beads but the number of phagocytosing Ezh2-/- cells was reduced. The average number of beads ingested by each cell was lower for Ezh2-/- cells compared to WT (5.95 vs 2.94, p < 0.001) in resting cells, and 9.47 vs. 3.73 in fMLP-activated cells, p < 0.01. The fraction of Ezh2-/- neutrophils generating ROS when stimulated with PMA is 2.4-fold higher than for WT cells. ROS production was greatly reduced in the presence of diphenyleneiodonium (DPI), confirming the role of NADPH oxidase in the generation of ROS. Conclusion Our results indicate impaired function of neutrophils derived from Ezh2-/- mice, demonstrating increased spontaneous cell death, impaired migration, decreased phagocytosis, and overproduction of ROS. Qualitative defects observed in neutrophils deficient for EZH2 may help explain the adverse prognosis associated with these mutations in MDS patients. Disclosures: Bejar: Genoptix: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity’s Board of Directors or advisory committees.

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Methyljasmonate, An Inhibitor of Glycolytic Energy Production, Displays Pre-Clinical Activity against Multiple Myeloma Cells.

Blood ◽

10.1182/blood.v114.22.1741.1741 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1741-1741

Author(s):

Steffen Klippel ◽

Jana Jakubikova ◽

Jake Delmore ◽

Melissa G. Ooi ◽

Douglas McMillin ◽

...

Keyword(s):

Cell Cycle ◽

Multiple Myeloma ◽

Cell Death ◽

Cancer Cells ◽

Board Of Directors ◽

Cell Lines ◽

Research Funding ◽

Energy Production ◽

Advisory Committees ◽

Normal Cells

Abstract Abstract 1741 Poster Board I-767 Background In contrast to most normal cells, cancer cells typically produce energy predominantly by glycolysis as demonstrated by O. Warburg more than 50 years ago. Methyljasmonate (MJ), a hormone produced by plants in response to biotic & abiotic stresses such as herbivory and wounding, has been shown to prevent the interaction of hexokinase (Hxk) and voltage dependent anion channels (VDACs), thereby significantly impacting the onset of glycolytic energy production. This may explain promising preclinical results observed with MJ against a variety of cancer cells, including myeloid leukemia and B-cell lymphoma cell lines. Methods and Results We tested the potential of MJ against Multiple Myeloma (MM) cells. We first evaluated the response of 16 different MM cell lines to 24 h of exposure to MJ concentrations of 0.5 – 3.5 mM using MTT assays. 15/16 of the MM cell lines tested displayed an IC50 of < 1.5 mM. In contrast, HS-5 stroma cells and peripheral blood mononuclear cells (PBMCs) did not respond to that MJ concentration, and even at a concentration of 2.5 mM MJ showed a maximal reduction of cell viability of 40%. Similarly to MM cell lines, purified CD138+ primary tumor cells of 3 MM patients displayed an IC50 of < 1.5 mM, suggesting that the differential sensitivity of MM vs. normal cells to MJ is not restricted to cell lines, but is also observed with primary tumor cells. Importantly, neither co-culture with HS-5 stroma nor IL-6 protected MM cells against MJ. Cell death commitment assays revealed that 1h exposure of 1.5 mM MJ induced cell death. Annexin V/PI FACS analysis of MJ-exposed MM cells showed that the cell death is mainly driven by apoptosis, evidenced by cleavage of caspases 3, 8 and 9 as well as of PARP. However, pre-incubation of MM cells with specific caspase inhibitors such as 10 mM of AC-DEVD-CHO, Z-IETD-fmk, Z-LEHD-fmk or 50 mM of Z-VAD only minimally protects the cancer cells from MJ exposure. Therefore, the impact of the MJ is not solely due to caspase triggered proteolytic cascades. Measurements of cellular ATP content by cell titer glow (CTG; Promega, Madison, WI) assay showed rapid depletion of ATP triggered by MJ action in sensitive MM cell lines. Additionally, we observed that 1 h exposure to 2 mM MJ modulated signaling pathways including IRS1/PI3K/AKT, MEK1/2, as well as Stat3 and JNK. FACS-based cell cycle analysis after propidium iodide staining did not show cell cycle arrest, but rather a rapid transition of cells to G0/G1 No correlation of sensitivity of MM cell lines and the number of mitochondria per cancer cell, as determined by Mitotracker Green (Invitrogen, Carlsbad, CA) -based flow analysis, was observed. We next examined if MJ exhibits either significant antagonism or synergy with established or novel anti-MM agents, including Bortezomib, Lenalidomide, Doxorubicin, Rapamycin or Dexamethasone, but discovered neither. However, MJ displayed synergy when combined with 2-Deoxyglucose. Finally, MJ was tested in vivo in scid/nod mice irradiated with 150 rads, injected with 1× 106 MM1S cells, and then, treated at 500 mg/kg by IP administration on a 5 days on / 2 days off schedule starting two weeks after tumor cell injection, There was an overall survival advantage of MJ-treated animals over the respective controls, with all treated mice (n=10) still alive but 6/10 control mice dead after 27 d. Conclusions Based on its rapidity of anti-MM action, favorable safety profile in preclinical models, distinct pattern of molecular sequelae, and compatibility with established anti-MM agents, MJ represents a promising investigational anti-MM agent. Disclosures Laubach: Novartis: Consultancy, Honoraria. Richardson:Millennium: (Speakers Bureau up to 7/1/09), Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: (Speakers Bureau up to 7/1/09), Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Anderson:Millennium: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Mitsiades:Novartis Pharmaceuticals: Consultancy, Honoraria; Milllennium: Consultancy, Honoraria; Bristol-Myers Squibb : Consultancy, Honoraria; Merck &Co.: Consultancy, Honoraria; Kosan Pharmaceuticals : Consultancy, Honoraria; Pharmion: Consultancy, Honoraria; PharmaMar: Patents & Royalties; Amgen: Research Funding; AVEO Pharma: Research Funding; EMD Serono: Research Funding; Sunesis Pharmaceuticals: Research Funding.

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Role of TORC1 and TORC2 in Multiple Myeloma

Blood ◽

10.1182/blood.v118.21.1815.1815 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1815-1815

Author(s):

Patricia Maiso ◽

Yi Liu ◽

Abdel Kareem Azab ◽

Brittany Morgan ◽

Feda Azab ◽

...

Keyword(s):

Cell Cycle ◽

Bone Marrow ◽

Board Of Directors ◽

Cell Lines ◽

Stromal Cells ◽

Research Funding ◽

Plasma Cells ◽

Advisory Committees ◽

Cycle Arrest

Abstract Abstract 1815 Mammalian target of rapamycin (mTOR) is a downstream serine/threonine kinase of the PI3K/Akt pathway that integrates signals from the tumor microenvironment. Mechanistically, mTOR operates in two distinct multi-protein complexes, TORC1 (Raptor) and TORC2 (Rictor). TORC1 leads to the phosphorylation of p70S6 kinase and 4E- BP1, while TORC2 regulates phosphorylation of Akt and other kinases. In multiple myeloma (MM), PI3K/Akt plays an essential role enhancing cell growth and survival and is activated by the loss of the tumor suppressor gene PTEN and by the bone marrow microenvironment. Rapamycin and its analogues have not shown significant activity in MM, likely due to the lack of inhibition of TORC2. In this study, we dissected the baseline activity of the PI3K/Akt/mTOR pathway TORC1/2 in MM cell lines with different genetic abnormalities. Methods: Eight different MM cell lines and BM samples from MM patients were used in the study. The mechanism of action was investigated by MTT, Annexin V, cell cycle analysis, immunochemistry, Western-blotting and siRNA assays. For the in vivo analyses, Luc+/GFP+ MM.1S cells (2 × 106/mouse) were injected into the tail vein of 30 SCID mice and tumor progression was detected by bioluminescence imaging. In vivo homing was checked by in vivo flow. Nanofluidic proteomic immunoassays were performed in selected tumors. Results: Raptor (TORC1) and Rictor (TORC2) knockdowns led to significant inhibition of proliferation of MM cells even in the presence of bone marrow stromal cells, this effect was also accompanied by inactivation of p-Akt, p-rS6 and p-4EBP1. We used INK128, a dual and selective TORC1/2 kinase inhibitor with similar effects to Raptor plus Rictor knockdown. We examined the protein expression levels of both mTOR complex and their downstream effectors in MM plasma cells from patients and cell lines. mTOR, Akt, pS6R and 4E-BP1 are constitutively activated in all samples. We showed that dual TORC1/2 inhibition is much more active than TORC1 inhibition alone (rapamycin) even in the presence of cytokines or stromal cells. INK128 induced cell cycle arrest, autophagy and apoptosis in cell lines and primary plasma cells even in the presence of bone marrow stromal cells (BMSCs). INK128 also showed a significant effect inhibiting cell adhesion in our in vivo homing model. Oral daily treatment with INK128 highly decreased the percentage of CD138+ tumor plasma cells in mice implanted with MM cells and reduced the levels of p-Akt and p-4EBP. These results suggest that potent and complete blockade of mTOR as part of TORC1 and TORC2 is potential therapeutic strategy to induce cell cycle arrest, apoptosis and disruption of MM cells interaction with the BM microenvironment. Conclusion: Dual inhibition of TORC1 and TORC2 represent a new and promising approach in the treatment of MM and its microenvironment. The ability of INK128 to inhibit both TORC1 and TORC2 strongly supports the potential use of this compound in MM patients. Disclosures: Liu: Intellikine: Employment. Roccaro:Roche: Research Funding. Rommel:Intellikine: Employment. Ghobrial:Celgene: Consultancy; Millennium: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Research Funding; Noxxon: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

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Targeting Hypersumoylation in Mantle Cell Lymphoma

Blood ◽

10.1182/blood-2019-127363 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4060-4060 ◽

Cited By ~ 1

Author(s):

Walter Hanel ◽

Liudmyla Tsyba ◽

Dennis Huszar ◽

Alex Prouty ◽

Xiaoli Zhang ◽

...

Keyword(s):

Cell Cycle ◽

Cell Death ◽

Board Of Directors ◽

Cell Lines ◽

Research Funding ◽

Advisory Committees ◽

Human B Cells ◽

Mantle Cell ◽

Normal Human ◽

Protein Sumoylation

Mantle cell lymphoma (MCL) is an aggressive and incurable subtype of B-cell Non-Hodgkin's lymphoma (NHL) characterized by genetic dysregulation of CyclinD1. Despite the improvement in response rates with current therapies, MCL patients inevitably relapse and outcomes remain poor. This is particularly true for MCL patients progressing on novel targeted therapies such as ibrutinib, highlighting the continued need for new therapeutic approaches. SUMOylation is a post-translational modification regulated by SUMO Activating Enzymes 1 and 2 (SAE1/2) affecting function, stability, and subcellular localization of a multitude of proteins such as Cyclin D1 and regulating multiple cellular functions such as cell cycle control and DNA damage response. While not yet explored in MCL, it is known that hyper-SUMOylation is associated with augmented cell proliferation and tumor growth of a number of cancers including B-cell NHL. We evaluated the expression levels of SAE1/2, total SUMO1, and SUMO 2/3 in normal human B cells, primary MCL patient samples, and a panel of 8 MCL cell lines via immunoblotting. We found significantly increased levels of SAE1/2 and total protein SUMOylation in 4 out of 5 MCL patient samples and all MCL cell lines compared to normal human B-cells. To validate the SAE complex as a potential therapeutic target in MCL, we performed genetic knockdown of SAE1 and SAE2 using both shRNA and an inducible CRISPR/Cas9 system and found significant reduction in viability of MCL cells (p < 0.001) thus confirming that SUMOylation is essential for MCL survival. TAK-981 (Takeda Pharmaceuticals) is a potent and selective inhibitor of the SAE1/2 complex currently in a phase 1 clinical trial (NCT036483). We found that treatment of MCL cell lines with TAK-981 resulted in time- and dose-dependent cell death in 7 of 8 MCL cell lines (IC50 17 - 62.5 nM at 72 hr) which was associated with relevant decrease in protein sumoylation. MCL cells were sensitive to TAK regardless of ATM or p53 mutations. Finally, TAK-981 treatment prolonged the survival of SCID mice engrafted with a human MCL cell line (Jeko) compared with placebo control [median overall survival (OS): TAK-981, 34 days; placebo, 29 days, p = 0.008] and also extended the survival of a novel patient derived xenograft (PDX) mouse model of ibrutinib-resistant MCL (median OS: TAK-981, 60 days; placebo, 55 days, p = 0.001), thus establishing the in vivo efficacy of TAK-981 in models of aggressive MCL. Mechanistically, 24 hours of treatment with TAK-981 resulted in a profound G2M cell cycle arrest in 6 out of 7 TAK-981-sensitive MCL cell lines. Cell synchronization with palbociclib followed by release into TAK-981 showed significant apoptosis upon G2M re-entry. In addition, in p53-deficient MCL cell lines, we found rapid accumulation of polyploid and aneuploid cells followed by rapid cell death following 48 hours of drug exposure. These findings strongly support mitotic catastrophe as a significant mechanism of tumor cell death mediated by TAK-981. Upon fractionation of cells at distinct phases of the cell cycle, we found significantly increased levels of protein SUMOylation by both SUMO1 and SUMO2/3 at the G2M transition. Further mechanistic data will be presented at the meeting. Given the multiple immune dampening mechanisms of SUMOylation, we are currently studying the anti-MCL immune effects of TAK-981. To do this, we are employing a novel immunocompetent mouse model of MCL in which murine lymphoma cells from Eμ-SOX11/CCND1 double transgenic animals are adoptively transferred into syngeneic mice. These mice develop a systemic lymphoma with morphological, molecular, and phenotypic features characteristic of MCL resulting in death within 3-4 weeks. Preliminary results with this model show that treatment with TAK-981 leads to decrease in lymphoma burden and significant prolongation of survival. Studies into the immune mediated anti-lymphoma effects of TAK-981 using this model are ongoing and will be presented at the meeting. Together, our data strongly support further development of TAK-981 as a novel MCL therapeutic. Disclosures Huszar: Takeda Pharmaceuticals: Employment, Equity Ownership. Parekh:Karyopharm Inc.: Research Funding; Foundation Medicine Inc.: Consultancy; Celgene Corporation: Research Funding. Maddocks:BMS: Research Funding; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Research Funding; Merck: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Teva: Membership on an entity's Board of Directors or advisory committees. Baiocchi:Prelude: Consultancy.

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Indirubins: A Potential Therapeutic Target in Multiple Myeloma

Blood ◽

10.1182/blood.v128.22.3259.3259 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3259-3259

Author(s):

Tina Bagratuni ◽

Nicolas Gaboriad-Kolar ◽

Roubini Zakopoulou ◽

Vassilios Myrianthopoulos ◽

Efstathios Kastritis ◽

...

Keyword(s):

Cell Cycle ◽

Multiple Myeloma ◽

Cell Death ◽

Virtual Screening ◽

Cell Line ◽

Board Of Directors ◽

Signaling Pathway ◽

Cell Lines ◽

Inhibitory Effects ◽

Advisory Committees

Abstract Current drugs in the treatment of Multiple Myeloma (MM) result in cell death via a number of mechanisms including a direct effect on plasma cells as well as alteration in the BM microenvironment. Although effective to some extent, none of the drug mechanisms of action are fully targeting a biological process essential and necessary for PC survival. Among the FDA approved kinase inhibitors, few are based on natural scaffolds. 6-bromoindirubin-3'-oxime (6BIO) is a potent kinase inhibitor based on the natural 6-bromoindirubin scaffold. Indirubin and 6-bromoindirubin are two natural products that have found a particular interest in dye chemistry as the main constituent of indigo and Tyrian purple dyes. Recent findings discovered that 6BIO was a promising anti-cancer agent acting on the JAK/STAT signaling pathway mediating cell proliferation. After enhancement of the chemical structure of 6BIO, further reports exposed that MLS-2438 and MLS-2384 were Akt signaling pathway inhibitor (MLS-2438) and potent c-Src kinase direct inhibitors. A library containing 2000 natural molecules was constructed using several data platforms. Each molecule was processed through different filters such as tautomeric studies, protonation and steroisomerism status in order to be used for calculations of virtual evaluation. Two approaches were followed: the structure-based virtual screening and the ligand-based virtual screening. To achieve structural based virtual screening, binding and evaluation of the chemical relation of each molecule in the crystallographic structure of the proteasome β5 subunit was performed. In the ligand-based virtual screening, calculations were made to identify the structural similarities of each molecule with the known proteasome inhibitor, bortezomib. The results of both approaches were combined, the molecules ranked, and 100 out of 2000 were identified as strong potential bioactive hits for the β5 subunit. Out of these 100 molecules, the chemical structures of high interest were the following: indole alkaloids derivatives (indirubins), flavonoids, secoiridoids, simple phenolic acids and acetophenone. A rational selection of indirubins derivatives was conducted in order to study their cytotoxic effects on MM. Fifty indirubins derivatives were selected based on different criteria: structure, known/unknown targets, chemodiversity in substitution patterns. To explore the inhibitory effects of indirubins in MM, we performed the WST1 proliferation assay in three MM cell lines (H929, JJN3, L363). Initially, all the selected indirubins (~50 indirubins) were tested at 7.5μM in L363 cell line and proliferation results from the WST1 assay extracted after 24 hours of treatment. More than half of the indirubins tested displayed more than 50% reduction of the proliferation at 7.5μM. Interestingly, 10 out of the 50 indirubins tested reduced more than 80% proliferation after 24 hours. The most active indirubins were tested in H929 and JJN3 cell lines, where similar effects were seen after 24 hours of treatment. All tested indirubins acted in a dose-dependent manner. Based on our first set of data, we suggest that indirubins have significant anti-proliferative effects on MM cell lines. Among the most active indirubins, two molecules namely 805 and 673 emerged as attractive for further development. Compound 805 is an analog of MLS-2384 while compound 673 is an analog of MLS-2438. The latter derivative represents a promising candidate displaying an IC50below the micromolar range on H929 and JJN3 cells. To determine the kind of cell death caused by one of the most active indirubins, 673, cell cycle analysis was performed before and after treatment in H929 cell line. In particular changes in RNA expression of 84 genes key to cell cycle regulation were analyzed in H929 cell line. Our results show that among other genes, the ones which have a dramatic increase in their expression (>5 fold) are mainly involved in cell cycle arrest such as GADD45A, CDC34, TP53, CHEK1 and CHEK2. A more detailed analysis of the profiler array will be presented at the meeting. In conclusion, this is the first study to show the inhibitory effects of indirubins in MM. Further investigation of these compounds may offer a therapeutic advantage that would affect MM pathogenesis and treatment. Disclosures Kastritis: Amgen: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Genesis: Consultancy, Honoraria. Terpos:Amgen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Janssen: Consultancy, Honoraria. Dimopoulos:Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Genesis: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

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Simvastatin Induces Death of Multiple Myeloma Cell Lines

Journal of Investigative Medicine ◽

10.1136/jim-52-05-34 ◽

2004 ◽

Vol 52 (5) ◽

pp. 335-344 ◽

Cited By ~ 11

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.

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Targeting MM at the Nexus between Cell Cycle and Transcriptional Regulation Via CDK7 Inhibition

Blood ◽

10.1182/blood-2020-142592 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 1-2

Author(s):

Yao Yao ◽

Woojun D Park ◽

Eugenio Morelli ◽

Mehmet Kemal Samur ◽

Nicholas P Kwiatkowski ◽

...

Keyword(s):

Cell Cycle ◽

Cell Proliferation ◽

Board Of Directors ◽

Cell Lines ◽

Mononuclear Cells ◽

Luciferase Reporter ◽

G1 Arrest ◽

Normal Donor ◽

Private Company ◽

Advisory Committees

Deregulated transcription and cell cycle control are hallmarks of cancer that are especially frequent in multiple myeloma (MM). Largely non-overlapping sets of cyclin-dependent kinases (CDKs) regulate cell division and RNA polymerase II (Pol II)-dependent transcription; and targeting of cell cycle CDKs has been long pursued as an attractive therapeutic strategy. Among CDKs, CDK7 presents a unique therapeutic opportunity as it functions as a CDK activating kinase (CAK), licensing the activity of cell cycle CDKs, and also serves as a core component of the general transcription factor TFIIH. Here we elucidated the biological role of CDK7 and its transcriptional regulatory landscape in MM, using genetic as well chemical approaches, including tools for CDK7 rapid protein degradation (dTAG) and the selective covalent inhibitor YKL-5-124 that targets a cysteine residue (C312) located outside of the kinase domain. We have observed that CDK7 inhibition via YKL-5-124 robustly inhibited the phosphorylation of the CDK1, 2 and 4 activation loops in a representative panel of MM cell lines at concentrations as low as 50 nM. This reduction was not observed in MM cells expressing a resistant mutation in the reactive cysteine (C312S). Consistent with decrease of CAK activity, we observed G1 arrest and S phase loss after CDK7 inhibition, which was also associated with a rapid and transient loss of Ser2 and Ser5 phosphorylation of the RNA Pol2 C-terminal domain. To understand the effect of CDK7 inhibition on MM cell growth and viability, we evaluated activity of YKL-5-124 across a large panel of 25 MM cell lines and observed a significant inhibition of MM cell proliferation, with a significantly lower IC50 compared to PHA-activated normal donor peripheral blood mononuclear cells (PBMCs), suggesting a specific sensitivity of MM cells to CDK7 inhibition. Longer exposure to YKL-5-124 caused apoptotic cell death in MM cells; however treatment with an inactive analog or in cells expressing the C312S mutation failed to inhibit MM cell proliferation, confirming that the antiproliferative potency of YKL-5-124 resides in its unique characteristic to covalently bind to C312 domain. Importantly, CDK7 inhibition impaired primary MM cells proliferation alone and when cultured in the presence of BM microenvironment. Selective pharmacological degradation of endogenously tagged CDK7 confirmed impact of CDK7 inhibition on MM cell proliferation via inhibition of CDK7 transcriptional and cell cycle activities. To complement the pharmacological studies, we have established MM cells to express inducible CRISPR/Cas9 constructs encoding 4 independent small guide RNAs targeting CDK7, resulting in the reduction of the abundance of CDK7 protein by 20-60% which was sufficient to inhibit MM cell viability over time, phenocopying pharmacologic inhibition of CDK7. These results support the view that CDK7 is a pharmacologically relevant target for MM. Gene expression analysis after CDK7 inhibition in MM1S and H929 cells revealed that transcripts for only a subset of genes were substantially affected by treatment with low dose of YKL-5-124, showing a strong leading-edge enrichment for downregulation of E2F expression program, cell cycle, DNA damage, and MYC targets. We have indeed confirmed a potent reduction in phosphorylation of RB protein, with consequent decrease of E2F activity in MM cells confirmed using E2F-driven luciferase reporter. These data suggest significant role for CDK7 in the CDK-pRB-E2F pathway in MM, which was strengthened by the observation of a positive correlation between expression of CDK7 and expression of E2F target genes in primary MM cells (n=409). Finally, we have evaluated the in vivo effect of CDK7 inhibition in several murine models of human MM. In the localized subcutaneous model, and the disseminated MM model where treatment with YKL-5-124 decreased tumor burden and improved survival. The effect of CDK7 inhibition explored in an aggressive, genetically engineered model of Myc-dependent MM, revealed evidence of response by decline in measurement of monotypic serum immunoglobulins. In conclusion, our study demonstrates that CDK7 contributes to the 'transcriptional addiction' and the cell cycle deregulation frequently observed in MM and represents an attractive molecular vulnerability to be exploited therapeutically. Disclosures Anderson: Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Oncopep and C4 Therapeutics.: Other: Scientific Founder of Oncopep and C4 Therapeutics.; Celgene: Membership on an entity's Board of Directors or advisory committees. Munshi:Takeda: Consultancy; Karyopharm: Consultancy; AbbVie: Consultancy; Amgen: Consultancy; Legend: Consultancy; Adaptive: Consultancy; Janssen: Consultancy; C4: Current equity holder in private company; OncoPep: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; BMS: Consultancy. Fulciniti:NIH: Research Funding.

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Cyperus articulatus L. (Cyperaceae) Rhizome Essential Oil Causes Cell Cycle Arrest in the G2/M Phase and Cell Death in HepG2 Cells and Inhibits the Development of Tumors in a Xenograft Model

Molecules ◽

10.3390/molecules25112687 ◽

2020 ◽

Vol 25 (11) ◽

pp. 2687

Author(s):

Mateus L. Nogueira ◽

Emilly J. S. P. de Lima ◽

Asenate A. X. Adrião ◽

Sheila S. Fontes ◽

Valdenizia R. Silva ◽

...

Keyword(s):

Cell Cycle ◽

Gas Chromatography ◽

Cell Death ◽

Liver Cancer ◽

Essential Oil ◽

Cell Cycle Arrest ◽

Cell Lines ◽

Hepg2 Cells ◽

Cycle Arrest

Cyperus articulatus L. (Cyperaceae), popularly known in Brazil as “priprioca” or “piriprioca”, is a tropical and subtropical plant used in popular medical practices to treat many diseases, including cancer. In this study, C. articulatus rhizome essential oil (EO), collected from the Brazilian Amazon rainforest, was addressed in relation to its chemical composition, induction of cell death in vitro and inhibition of tumor development in vivo, using human hepatocellular carcinoma HepG2 cells as a cell model. EO was obtained by hydrodistillation using a Clevenger-type apparatus and characterized qualitatively and quantitatively by gas chromatography coupled to mass spectrometry (GC-MS) and gas chromatography with flame ionization detection (GC-FID), respectively. The cytotoxic activity of EO was examined against five cancer cell lines (HepG2, HCT116, MCF-7, HL-60 and B16-F10) and one non-cancerous one (MRC-5) using the Alamar blue assay. Cell cycle distribution and cell death were investigated using flow cytometry in HepG2 cells treated with EO after 24, 48 and 72 h of incubation. The cells were also stained with May–Grunwald–Giemsa to analyze the morphological changes. The anti-liver-cancer activity of EO in vivo was evaluated in C.B-17 severe combined immunodeficient (SCID) mice with HepG2 cell xenografts. The main representative substances of this EO sample were muskatone (11.6%), cyclocolorenone (10.3%), α-pinene (8.26%), pogostol (6.36%), α-copaene (4.83%) and caryophyllene oxide (4.82%). EO showed IC50 values for cancer cell lines ranging from 28.5 µg/mL for HepG2 to >50 µg/mL for HCT116, and an IC50 value for non-cancerous of 46.0 µg/mL (MRC-5), showing selectivity indices below 2-fold for all cancer cells tested. HepG2 cells treated with EO showed cell cycle arrest at G2/M along with internucleosomal DNA fragmentation. The morphological alterations included cell shrinkage and chromatin condensation. Treatment with EO also increased the percentage of apoptotic-like cells. The in vivo tumor mass inhibition rates of EO were 46.5–50.0%. The results obtained indicate the anti-liver-cancer potential of C. articulatus rhizome EO.

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

Molecules ◽

10.3390/molecules25215016 ◽

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.

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Dual Targeting of -TORC1 and -TORC2 as a New Strategy In the Treatment of Multiple Myeloma

Blood ◽

10.1182/blood.v116.21.133.133 ◽

2010 ◽

Vol 116 (21) ◽

pp. 133-133 ◽

Cited By ~ 2

Author(s):

Patricia Maiso ◽

AbdelKareem Azab ◽

Yang Liu ◽

Yong Zhang ◽

Feda Azab ◽

...

Keyword(s):

Cell Cycle ◽

Multiple Myeloma ◽

Bone Marrow ◽

Board Of Directors ◽

Cell Lines ◽

Mechanism Of Action ◽

Plasma Cells ◽

Bone Marrow Microenvironment ◽

Advisory Committees

Abstract Abstract 133 Introduction: Mammalian target of rapamycin (mTOR) is a downstream serine/threonine kinase of the PI3K/Akt pathway that integrates signals from the tumor microenvironment such as cytokines and growth factors, nutrients and stresses to regulate multiple cellular processes, including translation, autophagy, metabolism, growth, motility and survival. Mechanistically, mTOR operates in two distinct multi-protein complexes, TORC1 and TORC2. Activation of TORC1 leads to the phosphorylation of p70S6 kinase and 4E-BP1, while activation of TORC2 regulates phosphorylation of Akt and other AGC kinases. In multiple myeloma (MM), PI3K/Akt plays an essential role enhancing cell growth and survival and is activated by the loss of the tumor suppressor gene PTEN and by the bone marrow microenvironment. Rapamycin analogues such as RAD001 and CCI-779 have been tested in clinical trials in MM. Their efficacy as single agents is modest, but when used in combination, they show higher responses. However, total inhibition of Akt and 4E-BP1 signaling requires inactivation of both complexes TORC1 and TORC2. Consequently, there is a need for novel inhibitors that can target mTOR in both signaling complexes. In this study we have evaluated the role of TORC1 and TORC2 in MM and the activity and mechanism of action of INK128, a novel, potent, selective and orally active small molecule TORC1/2 kinase inhibitor. Methods: Nine different MM cell lines and BM samples from MM patients were used in the study. The mechanism of action was investigated by MTT, Annexin V, cell cycle analysis, Western-blotting and siRNA assays. For the in vivo analyses, Luc+/GFP+ MM.1S cells (2 × 106/mouse) were injected into the tail vein of 30 SCID mice and tumor progression was detected by bioluminescence imaging. Nanofluidic proteomic immunoassays were performed in selected tumors. Results: To examine activation of the mTOR pathway in MM, we performed kinase activity assays and protein analyses of mTOR complexes and its downstream targets in nine MM cell lines. We found mTOR, Akt, pS6R and 4E-BP1 are constitutively activated in all cell lines tested independently of the status of Deptor, PTEN, and PI3K. All cell lines expressed either Raptor, Rictor or both; excepting H929 and U266LR7 which were negative for both of them. Moreover, primary plasma cells from several MM patients highly expressed pS6R while normal cells were negative for this protein. We found that INK128 and rapamycin effectively suppressed phosphorylation of p6SR, but only INK128 was able to decrease phosphorylation of 4E-BP1. We observed that INK128 fully suppressed cell viability in a dose and time dependent manner, but rapamycin reached a plateau in efficacy at ± 60%. The IC50 of INK128 was in the range of 7.5–30 nM in the eight cell lines tested. Similar results were observed in freshly isolated plasma cells from MM patients. Besides the induction of apoptosis and cell cycle arrest, INK128 was more potent than rapamycin to induce autophagy, and only INK128 was able to induce PARP and Caspases 3, 8 and 9 cleavage. In the bone marrow microenvironment context, INK128 inhibited the proliferation of MM cells and decreased the p4E-BP1 induction. Importantly, treatment with rapamycin under such conditions did not affect cell proliferation. INK128 also showed a significantly greater effect inhibiting cell adhesion to fibronectin OPM2 MM1S, BMSCs and HUVECs compared to rapamycin. These results were confirmed in vivo. Oral daily treatment of NK128 (1.0 mg/kg) decreased tumor growth and improved survival of mice implanted with MM1S. Conclusion: Dual inhibition of TORC1 and TORC2 represent a new and promising approach in the treatment of MM and its microenvironment. The ability of INK128 to inhibit both TORC1 and TORC2 strongly supports the potential use of this compound in MM patients. Disclosures: Anderson: Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Ghobrial:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees.

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