Detection and Quantification of Cereblon Protein and mRNA in Multiple Myeloma Cell Lines and Primary CD138+multiple Myeloma Cells

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4043-4043
Author(s):  
Anita K Gandhi ◽  
Herve Avet-Loiseau ◽  
Michelle Waldman ◽  
Anjan Thakurta ◽  
Sharon L Aukerman ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Protein Expression ◽  
Western Blot ◽  
Cell Lines ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Full Length ◽  
Employment Equity ◽  
Equity Ownership ◽  
Protein Variants

Abstract Abstract 4043 Background: Cereblon (CRBN), a component of the DDB1-CUL4A-Roc1 ubiquitin ligase complex, has been identified as a target of the immunomodulatory agents thalidomide, lenalidomide, and pomalidomide (Lopez-Girona et al. Leukemia. 2012; Zhu et al. Blood. 2011; Ito et al. Science. 2010.). CRBN binding by these agents mediates their anti-proliferative effects in multiple myeloma (MM) cells (Lopez-Girona et al. Leukemia. 2012; Zhu et al. Blood. 2011). However, the role of CRBN quantification as a marker for disease responsiveness or resistance to these drugs remains to be fully defined. Furthermore, it is unclear whether measuring mRNA or protein expression is the best approach for development of a quantitative CRBN expression assay. In order to define the optimal assay approach, we have studied CRBN mRNA and protein expression in MM cell lines (n=20) and MM patient samples. Methods: CRBN isoform mapping was undertaken using a nested PCR approach and Sanger sequencing. Commercially available and newly generated rabbit anti-CRBN antibodies were characterized with recombinant human CRBN protein and MM cell line extracts via western blot analysis. Results: Our data show that in addition to the transcript for full length protein (GenBank Accession NM_016302.3), in MM cells there are at least 6 alternatively spliced isoforms of CRBN as depicted in Figure 1. Five of the 6 CRBN isoforms (CRBN-003, -004, -005, -006, and -007) contain novel splice junctions not previously described. In addition, 3 of the identified transcripts (CRBN-002, -003, and -005) contain in-frame ORFs, suggesting they encode variants of CRBN protein. Of note, exon 10, which contains a portion of the IMiD-binding domain, is not present in CRBN-002. The functional consequence of CRBN-002 remains to be elucidated, but may be a marker of drug resistance. In order to measure CRBN protein levels, we developed and characterized three rabbit monoclonal antibodies to CRBN including antibody CRBN65, which has the potential to discriminate between the different CRBN protein products, including CRBN-002 by western blot analysis. Additionally, we compared 8 commercially available CRBN antibodies. Western blot analysis of cell lines with commercial and newly developed antibodies identified full length protein at 51 kD. Most commercial antibodies also identified multiple bands of other sizes which may represent CRBN protein variants; however, many are likely non-specific bands as they are larger than full-length CRBN. Conclusion: We have identified novel splice variants of CRBN from MM cell lines and primary tumor samples. The structure of the isoforms and their potential ability to be translated into several protein variants of CRBN reflect the complex regulation of the CRBN gene. These data suggest that accurate quantification of CRBN mRNA level in clinical studies may require measurement of both full-length CRBN mRNA as well as other mRNA isoforms. Currently available primers and gene expression arrays are not capable of identifying and/or resolving the complex set of CRBN isoforms present in cells. These data also demonstrate that CRBN65 is a highly specific and sensitive antibody that could be used for detection of CRBN and its key variants. Taken together, our data emphasize the importance for developing standardized reagents and assays for both mRNA and protein level measurement of CRBN before using them as markers for clinical response or resistance. Disclosures: Gandhi: Celgene Corp: Employment, Equity Ownership. Waldman:Celgene Corp: Employment, Equity Ownership. Thakurta:Celgene Corp: Employment, Equity Ownership. Aukerman:Celgene Corp: Employment, Equity Ownership. Chen:Celgene Corp: Employment, Equity Ownership. Mendy:Celgene Corp.: Employment, Equity Ownership. Rychak:Celgene Corp: Employment, Equity Ownership. Miller:Celgene Corp: Employment, Equity Ownership. Gaidarova:Celgene Corp: Employment, Equity Ownership. Gonzales:Celgene Corp: Employment, Equity Ownership. Cathers:Celgene Corp: Employment, Equity Ownership. Schafer:Celgene: Employment, Equity Ownership. Daniel:Celgene Corporation: Employment. Lopez-Girona:Celgene Corp: Employment, Equity Ownership. Chopra:Celgene Corp: Employment, Equity Ownership.

Inhibition Of H3K27-Methylome As a Novel Therapeutic Strategy In Multiple Myeloma

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3162-3162
Author(s):  
Maria Gkotzamanidou ◽  
Masood A Shammas ◽  
Jun Qi ◽  
Mehmet Kemal Samur ◽  
Teru Hideshima ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Western Blot ◽  
Cell Lines ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Inhibitory Effect ◽  
Epigenetic Changes ◽  
Healthy Donors ◽  
Equity Ownership ◽  
Induced Apoptosis

Abstract Multiple myeloma (MM) remains incurable, despite recent therapeutic advances; newer insights into the pathogenic mechanisms that cause this disease and additional therapies are urgently needed. Recent studies of the epigenome and in particular the methylome, have shown that myeloma is characterized by widespread epigenetic changes. Epigenetic changes may precede genetic mutations and genomic instability; Ubiquitously Transcribed Tetratricopeptide Repeat Protein X (UTX), a histone H3K27 demethylase may represent such an example. Previous studies have shown inactivating somatic mutations in UTX (KDM6A) in 10% of MM cases. Aberrant methylation of core histone tails and deregulation of the corresponding enzymes such as UTX and JMJD3 have been implicated in leukemia as well as other types of cancers, but their role in MM remains unknown. We evaluated the activity of the selective Jumonji H3K27 demethylase (UTX/JMJD3) inhibitor, GSK-J4, in MM. In a panel of 15 human MM cell lines (HMCLs) including cell lines resistant to bortezomib and dexamethasone, GSK-J4 induced significant survival and proliferation inhibition, as measured by luminescence-based viability assay (CTG), MTT (inhibition>68% in 48 hours) and 3H thymidine uptake, with the exception of OPM2 that was resistant up to 5 uM concentration. GSK-J4 induced apoptosis as measured by flow cytometry upon staining with Annexin-V/Propidium Iodide. The compound did not induce cytotoxicity in PBMCs from healthy donors and normal human skin fibroblasts. The inhibitory effect of GSK-J4 was observed also in CD138+ primary plasma cells from newly diagnosed MM patients (n=5) compared to PBMCs from healthy donors (n=9) (p<0.001). The compound also resulted in significant inhibition of MM cell colony formation in soft agar after 2 weeks in compared to controls. Moreover, interaction between MM cells and bone marrow stromal cells from MM patients or HS-5 stromal cell line did not overcome the inhibitory effect of GSK-J4 in all the HMCLs (KMS-12BM, LP-1, MM1S, OPM2, RPMI-8226, H929 and INA6) tested. To further investigate the mechanism of GSK-J4-induced apoptosis, we evaluate the activation of caspases 3/7, 8 and 9, and observed significant activation of caspase 3/7 and 9, indicating the involvement of intrinsic apoptotic pathway into the GSK-J4 function, as also confirmed by western blot analysis of Bcl-xl, p53, and Bax. We evaluated the enzymatic activity of UTX/JMJD3 in HMCLs by using a fluometric-based assay. The sensitivity of the HMCLs to demethylase inhibitor was directly related with UTX/JMJD3 activity (p<0.05). As the methylation of H3K27 mark plays a major role in the maintenance of active and silent states of gene expression in important cellular processes, we next mapped H3K27me3 and H3K27me2 chromatin modifications by genome-wide chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq) analysis in RPMI8226 and KMS-12BM cells before and after GSK-J4 treatment and in UTX knockdown cells. Transcription factors of OSKM complex: Oct4, Sox2, and Nanog were found to be targets of aberrant demethylation after treatment with GSK-J4, indicating possible involvement of H3K27 demethylases in pathogenesis of MM through the regulation of “stemness” genes. Western blot analysis showed that the inhibitor reduced the expression of these genes. In conclusion, we demonstrate a significant beneficial impact of inhibition of H3K27 demethylation in MM. These results provide new insights into the mechanism of altering methylome as a potent epigenetic intervention in treatment of MM. Disclosures: Hideshima: Acetylon Pharmaceuticals: Consultancy. Anderson:celgene: Consultancy; onyx: Consultancy; gilead: Consultancy; sanofi aventis: Consultancy; oncopep: Equity Ownership; acetylon: Equity Ownership.

scholarly journals Single Agent and Combinatorial Efficacy of First-in-Class Small Molecule ONC201 in Acute Leukemia and Multiple Myeloma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2759-2759 ◽  
Author(s):  
Varun V Prabhu ◽  
Amriti Lulla ◽  
Christina L Kline ◽  
Peter J Van den Heuvel ◽  
Mala K. Talekar ◽  
...  
Keyword(s):  
Western Blot ◽  
Cell Viability ◽  
Cell Lines ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Single Agent ◽  
Employment Equity ◽  
Equity Ownership ◽  
Dose Dependent Decrease ◽  
Dose Dependent

Abstract ONC201 is the founding member of the imipridone class of anti-cancer small molecules that possess a unique core chemical structure. ONC201 is currently being evaluated in several Phase I/II clinical trials for advanced cancers. In the current study, we evaluated the single agent and combinatorial efficacy of ONC201 in preclinical models of acute leukemia and multiple myeloma (MM). In acute leukemia, we evaluated ONC201 anti-cancer effects in acute myeloid leukemia (AML) (Kasumi-1, HL60) and acute lymphoblastic leukemia (ALL) (Reh, Jurkat and MOLT-4) cell lines. We observed a time- and dose-dependent decrease in cell viability for every cell line in the panel (EC50 1-5 µM). Vincristine-resistant cells HL60/VCR were also sensitive to single agent ONC201 with EC50 values on par with corresponding vincristine-sensitive parental cells. Dose- and time-dependent induction of apoptosis was noted in Western blot analysis of caspase-3 cleavage in AML cell lines treated with 2.5 µM or 5 µM of ONC201 for 48 hr. Western Blot analysis further demonstrated inhibition of Akt and Foxo3a phosphorylation in Kasumi-1 cells, in line with the previously reported late-stage signaling effects of ONC201 in solid tumor cells (Allen et al, 2013). Sub-G1 analysis indicated that ONC201 induces apoptosis in ALL cells and a pan-caspase inhibitor reduced ONC201-mediated apoptosis. Western blot analysis revealed ONC201-mediated apoptosis involves PARP cleavage and caspase-9 activation in ALL cells. Anti-apoptotic Bcl-2 family members Bcl-2 and Bcl-xl were downregulated while the pro-apoptotic Bcl-2 family member Bim is upregulated in response to ONC201 treatment in ALL cells. ONC201 also downregulates the inhibitor of apoptosis (IAP) family proteins cIAP1 and cIAP2 in ALL cells. We observed inhibition of Akt phosphorylation upon ONC201 treatment of ALL cells. Fresh AML patient cells were also found to be sensitive to ONC201 in cell viability and caspase 3/7 activity assays at 5µM. We observed that independent clones of cancer cells with acquired resistance to ONC201 were more sensitive to cytarabine compared to parental ONC201-sensitive cancer cells. In addition, ONC201 demonstrated synergistic reduction in cell viability in combination with cytarabine in AML cell lines. Determination of combination indices (CI) revealed synergy at several concentrations (CI 0.336-0.75 in CMK cells). Also, ONC201 combined additively with midostaurin in CMK cells and vincristine in HL60/VCR cells. Thus, ONC201 is a promising combinatorial partner for AML therapies based on these preclinical sensitization results. In accordance with ONC201-mediated activation of the integrated stress response that B cells are highly sensitive to (Kline et al and Ishizawa et al, 2016), MM was identified as one of the most ONC201-sensitive tumor types in the Genomics of Drug Sensitivity in Cancer collection of cell lines. Three human MM cell lines were used for validation (KMS18, MM.1S and RPMI-8226), which revealed a time- and dose-dependent decrease in cell viability (EC50 1-2.5 µM). Bortezomib-resistant cells MM.1S 33X were sensitive to ONC201 as a single agent with EC50 values comparable to bortezomib-sensitive parental cells. We observed an average of 10-fold induction of ONC201-mediated apoptosis using Sub-G1 analyses in MM cells at 5 µM, 48 hrs post-treatment. Rescue of ONC201-mediated apoptosis was demonstrated using the pan-caspase inhibitor (Z-VAD-FMK). In addition, Western blot analysis in MM cells indicated a dose-dependent decrease in the anti-apoptotic protein XIAP which is a key mediator of apoptosis inhibition and is reported to be highly up-regulated in MM cells. Furthermore, ONC201 demonstrated synergistic reduction in cell viability at various concentrations in combination with either ixazomib or dexamethasone, which are used in the clinical treatment of MM, in RPMI8226 cells (CI 0.228-0.75). Also, ONC201 combined additively with bortezomib in RPMI8226 and MM.1S 33X cells. In summary, these preclinical studies support the ongoing ONC201 single agent trials in acute leukemias and MM. Our findings suggest that ONC201 may be an important therapeutic option for patients with hematological malignancies who have developed resistance to approved therapies. Additionally, our results point to specific standard-of-care therapies that may be combined with ONC201 to exert durable responses without adding to the burden of toxicity. Disclosures Prabhu: Oncoceutics: Employment. Tarapore:Oncoceutics: Employment, Equity Ownership. Oster:Oncoceutics: Employment, Equity Ownership. Allen:Oncoceutics: Employment, Equity Ownership. El-Deiry:Oncoceutics: Equity Ownership.

BCL-2/BCL-XL Inhibition in Smoldering Multiple Myeloma CD138+ Cells: Functional Effects and Protein Expression Levels

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5159-5159
Author(s):  
Francesco Libotte ◽  
Maria Rosaria Ricciardi ◽  
Elisabetta Calabrese ◽  
Cinzia De Benedictis ◽  
Sara Santinelli ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Growth ◽  
Protein Expression ◽  
Western Blot ◽  
Cell Lines ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Plasma Cells ◽  
Family Members ◽  
Primary Cells

Abstract Smoldering multiple myeloma (SMM) is an asymptomatic plasma-cell disorder with a high risk of progression to a symptomatic multiple myeloma (MM). The time and mechanisms underlying the progression are still unclear. The Bcl-2 family proteins are frequently aberrantly expressed in MM and support proliferation, survival and drug-resistance. The activity of inhibitors of the anti-apoptotic Bcl-2 family members is currently under investigation in MM, while their therapeutic potential has never been explored in SMM. In this study, we investigated the constitutive protein expression profile of anti-apoptotic Bcl-2 family members and the functional effects of their pharmacological modulation by the Bcl-2/Bcl-xL inhibitor ABT-737 (kindly provided by Abbott Laboratories) on primary cells from SMM and symptomatic MM at different stages of the disease. Mononuclear cells isolated from BM aspirates of MM patients were fractionated into purified CD138+ (>80%) and CD138- populations. Protein extracts were prepared and subjected to Western blot analysis for anti-apoptotic Bcl-2 family members. Within the CD138+ malignant plasma cells, SMM samples were characterized by elevated Mcl-1 levels and barely detectable expression of Bcl-xL, while symptomatic MM samples exhibited higher levels of both Mcl-1 and Bcl-xL. No significant differences in Bcl-2 level expression were instead observed between SMM and symptomatic MM samples. We, then, investigated the functional effects of ABT-737 on MM cell line and on CD138+ malignant plasma cells from SMM and symptomatic MM samples. MM cell lines exposed to increasing concentrations of ABT-737 (10–1000 nM) for up 72 hours showed a dose- and time-dependent cell growth inhibition (IC50 200–400 nM at 72 hours) due to the loss of mitochondrial membrane potential, caspase activation and significant increase in the proportion of apoptotic cells. In addition, cell cycle analysis demonstrated a significant G1-phase depletion in KMS18 cells exposed to 1000 nM ABT-737 for 72 hours (26.9%±1.4 vs 54.6%±4.5, p=0.021). Western Blot analysis demonstrated that ABT-737 dramatically reduces Bcl-2 protein levels, with appearance of cleaved forms, while Mcl-1 was down-regulated only at higher doses. No effects were seen on Bcl-xL expression. The ABT-737 activity was then examined on CD138+ purified primary cells from 9 SMM samples cultured with ABT-737 (10–1000 nM) for 72 hours. A remarkable reduction in cell growth and increase in the percentage of apoptotic cells was observed in response to 100 nM ABT-737. In particular, a statistically significant pro-apoptotic activity of 100 nM ABT-737 was demonstrated by the increment of the subG1 peak after 24 hours (25.0%±9.8 vs 60.1%±17.0, p=0.00011, and vs 78.1%±14.0, p=0.0003, in the presence of 100 and 1000 nM ABT 737, respectively). Similar results were obtained in the CD138+ fraction from symptomatic MM (7 newly diagnosed and in 9 relapsed/refractory) with a significant increment of the subG1 peak after 24 hours of ABT-737 exposure (27.9%±15.5 vs 53.4%±16.8, p<0.0001, and vs 70.0%±15.4, p<0.0001, in the presence of 100 and 1000 nM ABT 737, respectively). Preliminary results indicate that, as in cell lines, Bcl-2 cleavage also occurs in CD138+ primary cells in response to ABT–737. In conclusion, we present the first evidence that Bcl-2/Bcl-xL inhibition by ABT-737 enhances apoptosis in CD138+ primary cells from SMM. We therefore suggest that this strategy may be developed for therapeutic purposes in different disease stages of MM patients.

scholarly journals Lenalidomide Promotes Degradation of Casein Kinase 1a (CK1a) through Cereblon: Implications for the Efficacy of Lenalidomide in MDS and AML

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3606-3606
Author(s):  
Paul Hollenbach ◽  
Ling Lu ◽  
Anita K. Gandhi ◽  
Rajesh Chopra ◽  
Kyle J MacBeth
Keyword(s):  
Western Blot ◽  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Cancer Cell ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Employment Equity ◽  
Protein Levels ◽  
Equity Ownership

Abstract Background: Lenalidomide has broad clinical activity in hematologic malignancies, including lymphoid malignancies (multiple myeloma (MM), non-Hodgkin’s lymphomas, B cell chronic lymphocytic leukemia) and myeloid malignancies (myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML)). Lenalidomide’s molecular mechanism involves modulation of the cullin 4 RING E3 ubiquitin ligase complex (CRL4-CRBN), with downstream effects on protein homeostasis. The binding of lenalidomide to CRL4-CRBN promotes ubiquitination and degradation of Aiolos and Ikaros in B cell lineages (MM, lymphoma), and regulation of Ikaros has been shown to be a key effector of lenalidomide’s anti-myeloma tumoricidal and T cell immunomodulatory activities. Multiple mechanisms of clinical efficacy have been hypothesized for lenalidomide activity in del(5q) MDS; however, lenalidomide-regulated substrates of therapeutic relevance to myeloid malignancies have not been defined. We undertook a proteomics approach to identify such substrates. Methods: Changes in global cellular protein levels were measured by tandem-mass-tagged proteomics in a del(5q) MDS cell line (MDS-L) and an AML cell line (HNT-34), following treatment with vehicle or 10 uM lenalidomide for 8, 24 and 72 hours. Western blot analysis was used to subsequently validate proteins that were differentially regulated in these lenalidomide-sensitive cell lines. Sensitivity to lenalidomide treatment in a panel of myeloid cancer cell lines was evaluated by tritiated thymidine and/or BrdU assays. Results: Across a panel of myeloid cancer cell lines evaluated for sensitivity to lenalidomide, HNT-34 and MDS-L cells were the most sensitive to lenalidomide, with EC50’s of 0.6 and 1.5 µM, respectively, while other cell lines were predominantly insensitive (EC50 > 10 µM). Proteomic analysis of lenalidomide-regulated proteins in MDS-L cells revealed Ikaros as the most down-regulated protein at 72 hrs (5.3 fold), and CK1α was the second most down-regulated protein (2.8 fold). The decrease of Ikaros and CK1α proteins by lenalidomide was confirmed by Western blot analysis in MDS-L and HNT-34 cells. Ikaros protein levels were reduced by lenalidomide in cell lines that were sensitive or insensitive to lenalidomide, suggesting that modulation of other proteins may be responsible for lenalidomide sensitivity of myeloid cancer cells. Ikaros levels were also decreased by a CRBN-binding glutarimide analog in HNT-34 cells, which were insensitive to the analog, further suggesting that Ikaros was not a substrate of consequence to sensitivity. In contrast, an initial study of five cell lines showed that lenalidomide promoted the greatest degradation of CK1a in the most sensitive lines (HNT-34, MDS-L), but did not degrade CK1a in the most insensitive line (MOLM-13). Mechanistic studies addressing CK1α regulation in HNT-34 cells revealed that CK1α protein levels were reduced by lenalidomide treatment in a time- and dose-dependent manner, with maximal reduction of 3.3 fold observed at 4 hrs using 10 µM lenalidomide, and CK1α degradation observed with lenalidomide at a dose as low as 0.1 µM. Pre-treating HNT-34 cells with the proteasome inhibitor MG-132 stabilized CK1α protein levels in the presence of lenalidomide, demonstrating proteasome-dependent degradation. A competition experiment performed by pre-treating HNT-34 cells with the glutarimide analog that binds CRBN, but does not result in the degradation of CK1a, resulted in CK1α protein stabilization in the presence of lenalidomide, demonstrating CRBN-dependence of the lenalidomide-induced degradation. Degradation of CK1α was also observed in primary peripheral blood mononuclear cells of AML patients treated with lenalidomide. Conclusions: CK1α is a lenalidomide-induced substrate of CRL4-CRBN, with initial links to myeloid cancer cell line sensitivity to lenalidomide. As the CSNK1A1 gene is located at 5q32, a commonly deleted region in MDS, further reduction of haplo-insufficient expression of CK1α is a potential mechanism of sensitivity to lenalidomide in del(5q) MDS. The therapeutic relevance of CK1α regulation by lenalidomide in AML requires further exploration. CK1α gene silencing and additional correlative studies of lenalidomide-induced degradation of CK1α are ongoing to define mechanistic links to myeloid cancer cell sensitivity to lenalidomide. Disclosures Hollenbach: Celgene: Employment, Equity Ownership. Lu:Celgene Corp: Employment. Gandhi:Celgene Corp: Employment, Equity Ownership. Chopra:Celgene Corp: Employment, Equity Ownership. MacBeth:Celgene: Employment, Equity Ownership.

Effect of the BET Inhibitor, Cpi-0610, Alone and in Combination with Lenalidomide in Multiple Myeloma

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4255-4255 ◽  
Author(s):  
Ka Tat Siu ◽  
Homare Eda ◽  
Loredana Santo ◽  
Janani Ramachandran ◽  
Miroslav Koulnis ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Protein Expression ◽  
Cell Lines ◽  
Positive Feedback ◽  
Research Funding ◽  
Cytotoxic Effect ◽  
Feedback Mechanism ◽  
Employment Equity ◽  
Equity Ownership ◽  
Rpmi 8226

Abstract The bromodomain and extraterminal (BET) proteins recognize acetylated lysine residues on histone tails and recruit transcriptional machinery to promote gene expression. The BET proteins are attractive drug targets because they regulate the expression of MYC, BCL2 and NF- κB target genes. We investigated the therapeutic potential of CPI-0610, an inhibitor of BET proteins, currently in Phase I testing in multiple myeloma (MM). Our preliminary data show that human MM cell lines are sensitive to BET inhibition, with IC50 values of 800-1000 nM being observed in MM.1S, MM.1R, RPMI-8226, LR5, H929 and U266 cell lines in 72h culture. We further show that CPI-0610 inhibits MM cell growth in the presence of cytokines and when co-cultured with bone marrow stromal cells. CPI-0610 induces apoptosis and G1 cell cycle arrest associated with MYC downregulation. However, protein levels of BCL2, NF- κ B and MCL1 remain unchanged in MM cells upon BET inhibition. The zinc finger transcriptional factor Ikaros (IKZF1) is highly expressed in MM (GEO dataset GSE36133). It is actively transcribed in the MM.1S cell line with an active transcription start site occupied by BRD4 and MED1 (Loven J et al. Cell 2013). Interestingly, we found that CPI-0610 suppresses Ikaros and IRF4 expression at the levels of both transcription and protein in MM cells. With the use of doxycycline-inducible shRNAs targeting IKZF1, IRF4 and MYC, we identified a positive feedback mechanism that is critical for MM cell survival. Individual knockdown of IRF4, IKZF1 or MYC all lead to induction of apoptosis in MM cells. Suppression of IRF4 decreases both Ikaros and MYC protein expression, suggesting that IRF4 is upstream of both Ikaros and MYC. Downregulation of MYC protein expression is observed following IKZF1 knockdown, suggesting that MYC is downstream of Ikaros. Finally, we observed a decrease in IRF4 protein level upon MYC downregulation, implicating a feedback mechanism from MYC to IRF4. Together, these data illustrate a molecular sequence of events going from IRF4 to IKZF1 to MYC and then back to IRF4, forming a positive feedback loop in MM cells. Based on the observation that shRNA-mediated knockdown of MYC and IKZF1 are toxic to MM, we combined CPI-0610 with lenalidomide, an immunomodulatory drug which stabilizes cereblon and facilitates Ikaros degradation in MM cells (Kronke J et al., and Lu G et al., Science 2014). We observed a synergistic cytotoxic effect in the cell lines tested (MM.1S and RPMI-8226). The enhanced cytotoxic effect of the combined treatment in MM cell lines is due in part to suppression of MYC, IKZF1 and IRF4. Ongoing studies will focus on understanding the molecular mechanism underlying this synergistic combination and validating its efficacy in vivo in order to provide a rationale for clinical protocols of BET inhibitors in MM. Disclosures Mertz: Constellation Pharmaceuticals: Employment, Equity Ownership. Sims:Constellation Pharmaceuticals: Employment, Equity Ownership. Cooper:Constellation Pharmaceuticals: Employment, Equity Ownership. Raje:Celgene Corporation: Consultancy; Eli Lilly: Research Funding; Takeda: Consultancy; Amgen: Consultancy; Onyx: Consultancy; AstraZeneca: Research Funding; Novartis: Consultancy; BMS: Consultancy; Acetylon: Research Funding; Millenium: Consultancy.

SIRT1 Activator 3 Potently Induces Apoptosis in Multiple Myeloma Cell Lines and in Primary Human Multiple Myeloma Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3845-3845
Author(s):  
Tommasina Guglielmelli ◽  
Emilia Giugliano ◽  
Ilaria Defilippi ◽  
Marisa Pautasso ◽  
Roberta Merlini ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Western Blot ◽  
Cell Lines ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Annexin V ◽  
Fixed Dose ◽  
Cell Leukaemia ◽  
Myeloma Cells ◽  
Sirt1 Activator

Abstract Abstract 3845 Poster Board III-781 Introduction Multiple myeloma (MM) is a malignant plasma cells disorder and as far as now remains an incurable disease so that new target therapies are necessary to improve survival of MM patients. Histone deacetylases (HDACs) are enzymes that deacetylate acetyl lysines in histones and various non-histone proteins. Class I and II HDACs have been identified as valid anticancer targets so that clinical studies on their inhibitors as new anticancer agents are ongoing. Much less is known about the consequences of inhibition and activation of the seven members of class III HDACs (sirtuins, SIRT1-7). Recent studies highlight the role of SIRT1 in stress response and cell survival. Increased levels of SIRT1 were observed in normal colon mucosa but not in advanced colon carcinomas. Moreover, SIRT1 activators have been shown to have anti-inflammatory property mediated by TNF-alpha. We have investigated the anti-myeloma activity of SIRT1 activator 3 in RPMI8226 and U226 MM cell lines and in 4 primary human MM cells. Methods In this study RPMI8226 and U226 MM cell lines were used. Bone marrow samples of 3 MM patients were subjected to CD138 immunomagnetic purification for plasmacells enrichment. Peripheral plasmacells from a patients with plasmacell leukaemia dexametasone-melphalan resistant were also collected. SIRT1 activator 3 was used at increasing dose of 50, 100 and 500 μM alone and in combination with 1 μM of dexametasone in MM cell lines and at the fixed dose of 500 μM in primary human plasmacells. Apoptosis has been assayed at 12h, 24h and 48h after treatment in RPMI8226 and U226 cells and at 24h in human plasmacells by flow cytometry evaluating annexin V marker. Western blot analysis was performed to assess the effect of SIRT1 activator 3 agent on NFkB activity (localization of p65 subunit), AKT, p-AKT, mTOR, p-mTOR, Src and p-Src. Results The highest level of apoptosis was observed in RPMI8226 cells with SIRT1 activator 3 agent at the dosage of 500 μM at 24 h. (annexin V positivity 53%, P<0.05). U226 cells resulted sensitive in the same conditions ((annexin V positivity 41%, P<0.05). SIRT1 activator 3 (500 μM) induced significant apoptosis at 24h (range 29-47%) in MM cells of all four patients. The highest level of apoptosis was observed in plasmacells of the patient with plasma cell leukaemia dexametasone-melphalan resistant (47% annexin V positivity) Dexametasone do not significantly enhances SIRT1 activator 3 induced apoptosis in both MM cell lines and primary human MM cells. Western blot analysis demonstrated strong reduction of AKT, mTOR and Src phosphorylation in RPMI8226 and U226 cells as early as 24h after exposure. Conclusion SIRT1 activator 3 induces significant cell death in MM cell lines and primary human myeloma cells in a dose-dependent manner. The mechanisms of SIRT1 activator 3 cytotoxicity are related to down-regulation of AKT, SRC and TOR phosphorylation. Together, these findings may give useful insights into a novel anti-myeloma therapy. Disclosures: Guglielmelli: Celgene: Honoraria; Janssen Cilag: Honoraria. Saglio:Celgene: Honoraria; Novartis: Honoraria; Bristol Myers: Honoraria.

Synthesis and Characterization of Quinoline-3-Carboxamide Derivatives as Inhibitors of the ATM Kinase

2020 ◽  
Vol 20 (23) ◽  
pp. 2070-2079
Author(s):  
Srimadhavi Ravi ◽  
Sugata Barui ◽  
Sivapriya Kirubakaran ◽  
Parul Duhan ◽  
Kaushik Bhowmik
Keyword(s):  
Western Blot ◽  
Cancer Cells ◽  
Cell Line ◽  
Cell Lines ◽  
Cancer Cell ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Cancer Cell Lines ◽  
Atm Kinase ◽  
Cytotoxicity Studies

Background: The importance of inhibiting the kinases of the DDR pathway for radiosensitizing cancer cells is well established. Cancer cells exploit these kinases for their survival, which leads to the development of resistance towards DNA damaging therapeutics. Objective: In this article, the focus is on targeting the key mediator of the DDR pathway, the ATM kinase. A new set of quinoline-3-carboxamides, as potential inhibitors of ATM, is reported. Methods: Quinoline-3-carboxamide derivatives were synthesized and cytotoxicity assay was performed to analyze the effect of molecules on different cancer cell lines like HCT116, MDA-MB-468, and MDA-MB-231. Results: Three of the synthesized compounds showed promising cytotoxicity towards a selected set of cancer cell lines. Western Blot analysis was also performed by pre-treating the cells with quercetin, a known ATM upregulator, by causing DNA double-strand breaks. SAR studies suggested the importance of the electron-donating nature of the R group for the molecule to be toxic. Finally, Western-Blot analysis confirmed the down-regulation of ATM in the cells. Additionally, the PTEN negative cell line, MDA-MB-468, was more sensitive towards the compounds in comparison with the PTEN positive cell line, MDA-MB-231. Cytotoxicity studies against 293T cells showed that the compounds were at least three times less toxic when compared with HCT116. Conclusion: In conclusion, these experiments will lay the groundwork for the evolution of potent and selective ATM inhibitors for the radio- and chemo-sensitization of cancer cells.

Abstract WP433: Targeting Organic Anion Transporting Polypeptide 1a4 (Oatp1a4) Expression at the Blood-brain Barrier: Implications for Treatment of Ischemic Stroke

Stroke ◽  
2017 ◽  
Vol 48 (suppl_1) ◽  
Author(s):  
Wazir Abdullahi ◽  
Hrvoje Brzica ◽  
Patrick Ronaldson
Keyword(s):  
Protein Expression ◽  
Western Blot ◽  
Rat Brain ◽  
Blood Brain Barrier ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Organic Anion ◽  
Stroke Treatment ◽  
Brain Microvessels ◽  
Cns Delivery

Introduction: Stroke is a leading cause of mortality and morbidity. Several drugs with neuroprotective properties have been proposed for stroke treatment but many have failed in clinical trials. These failures may be due to limited drug permeability across the blood-brain barrier (BBB). Targeting endogenous BBB uptake transporters (i.e., organic anion transporting polypeptides (Oatps)) is a novel approach that can improve CNS delivery of drugs relevant to stroke therapy (i.e., statins). Optimal CNS drug delivery via Oatp in the setting of stroke requires characterization of regulatory pathways such as transforming growth factor (TGF)-β signaling. The goal of the present study was to investigate, in vivo , involvement of TGF-β signaling via the activin-like kinase (ALK)-1 receptor on Oatp1a4 expression at the BBB. Methods: Female Sprague-Dawley rats (200-250 g) were administered bone morphogenic protein-9 (BMP-9; 0-5 μg/kg, i.p.), an ALK1 agonist, or vehicle (i.e., 0.9% saline). Inhibition experiments were performed using LDN193189 (0-5 mg/kg, i.p.), an ALK1 antagonist. Western blot analysis and fluorescence microscopy of isolated brain microvessels were used to determine protein expression and localization in rat brain microvessels respectively. Results: Fluorescence staining demonstrated localization of Oatp1a4 and ALK1 in rat brain microvessels. Western blot analysis showed a dose dependent increase in Oatp1a4 protein expression in brain microvessels isolated from BMP-9 treated rats as compared to controls. Treatment with 0.5 μg/kg and 5 μg/kg BMP-9 resulted in a 55% and 116% increase in Oatp1a4 protein expression, implying that activation of ALK1 signaling can up-regulate Oatp1a4 at the brain microvasculature. In contrast, 6 h treatment with LDN193189 did not alter Oatp1a4 expression across a dose range of 0-5 mg/kg, suggesting that ALK1 inhibition does not modulate basal Oatp1a4 expression at the BBB. Conclusions: Taken together, our data implies that TGF-β/ALK1 signaling may play a role in altering Oatp1a4 protein expression at the BBB. Studies are currently being undertaken in our laboratory to fully characterize the role of TGF-β/ALK1 signaling in determining CNS delivery of drugs relevant to stroke treatment (i.e., statins).

Abstract 234: Interleukin 2 Promotes Proliferation and Migration in Vascular Smooth Muscle Cells

2017 ◽  
Vol 37 (suppl_1) ◽  
Author(s):  
Spencer Barnhill ◽  
Prakash Arumugam ◽  
John Matsuura ◽  
Scott Berceli ◽  
Katie Carroll ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Protein Expression ◽  
Western Blot ◽  
Vascular Smooth Muscle Cells ◽  
Blot Analysis ◽  
Intimal Hyperplasia ◽  
Western Blot Analysis ◽  
Interleukin 2 ◽  
And Migration ◽  
Proliferation And Migration

Interleukin-2 (IL-2) is primarily known as a soluble cytokine that regulates T cell responses. We previously reported, however, that IL-2 is retained in the extracellular matrix by association with perlecan, a heparan sulfate proteoglycan (HSPG). Perlecan is the main HSPG in vascular basement membranes, and previous studies from our laboratory demonstrated that, in human arteries, vascular smooth muscle cells (VSMC) are surrounded by perlecan-bound IL-2. We also noted that IL-2 deficient mice lose SMCs with age, leading to widened esophagi and aortic aneurysms. Given this information, we hypothesized that IL-2 has a direct impact on VSMC, and that VSMC express functional IL-2 receptors (IL-2R). We therefore examined both protein and mRNA expression of each of the three IL-2R subunits (alpha, beta, gamma) on human VSMC grown from arterial explants. These VSMC expressed SMC actin, smooth muscle myosin heavy chain, and when quiescent, smoothelin. Protein expression was assessed by in cell Western and by Western blot analysis. Receptor expression was evaluated under distinct culture conditions, which yielded highly proliferative, intermediate, or quiescent VSMC. Contractile protein expression was low, intermediate, or high, respectively, consistent with the characteristics of proliferating vs quiescent SMCs. Each phenotype expressed all 3 subunits of the IL-2R. IL-2 subunits appeared to follow a cytoskeletal pattern in cells expressing high levels of contractile proteins. Western blot analysis of VSMC lysates revealed expression of all 3 receptors at molecular weights identical to lysates from a T cell line. VSMCs also expressed mRNA for each receptor subunit. Functionally, IL-2 promoted migration (using a Boyden chamber assay) and proliferation in a dose dependent fashion. Because excess proliferation and migration are critical to intimal hyperplasia, we asked whether IL-2 levels change under conditions known to generate intimal hyperplasia. In a rabbit model, IL-2 mRNA increased in venous grafts exposed to high flow for 2h. IL-2 levels, by Western blot, were also increased in human hyperplastic veins. In conclusion, these data show that VSMC have functional IL-2R, and suggest that IL-2 may contribute to the development of intimal hyperplasia.

scholarly journals Proteomic profiles of unilateral cryptorchidism in pigs at different ages using MALDI-TOF mass spectrometry and in-gel digestion coupled with mass spectrometry (GeLC-MS/MS) approaches

2020 ◽  
Vol 16 (1) ◽  
Author(s):  
Nathamon Yimpring ◽  
Sittiruk Roytrakul ◽  
Janthima Jaresitthikunchai ◽  
Narumon Phaonakrop ◽  
Sucheewin Krobthong ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Protein Expression ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Expression Profiles ◽  
Tumor Necrosis Factor Receptor ◽  
Principal Component ◽  
Peptide Mass ◽  
Different Ages

Abstract Background Cryptorchidism is a condition that occurs when one or both testes fail to descend into the scrotum. It is a common congenital disorder, causing economic loss in pig production. However, there have been only limited studies of differential protein expression profiles in undescended testes (UDTs) in the abdomen and descended testes (DTs) in cryptorchid pigs, especially at the peptidome and proteome levels. The present study aimed to analyze the peptidome of UDTs and DTs in unilateral cryptorchid pigs aged 1–2, 6, 15 and 20 weeks and in normal testes of healthy pigs aged 1–2 and 12 weeks, using peptide mass fingerprinting and three-dimensional principal component analysis (3D-PCA) with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and to identify potential protein candidates, using in-gel digestion coupled with mass spectrometry (GeLC-MS/MS). Western blot analysis was used to verify protein expression. Protein sequence was affirmed by liquid chromatography–tandem mass spectrometry. Results A PCA plot showed a discrete cluster for each sample group. Peptide mass fingerprints (PMFs) demonstrated unique peptide fragments in UDTs at different ages. A number of markedly expressed proteins from GeLC-MS/MS were identified, including the multifunctional tumor necrosis factor receptor superfamily member 18 (TNFRSF18), in DTs at 1–2 and 6 weeks and in UDTs at 15 and 20 weeks of age. Using western blot analysis, high expression of TNFRSF18 was observed in the UDTs at 15 weeks. Using the STITCH database, this protein was found to be related to apoptosis, corresponding to the previous report in the UDTs at the same age. Conclusions The present study revealed the specific PMFs and clusters for porcine cryptorchidism, and a novel protein, TNFRSF18, associated with the disease mechanism. These results could provide further insights into the pathogenesis of the disease.

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