Chk1 inhibition potently blocks STAT3 tyrosine705 phosphorylation, DNA binding activity, and activation of downstream targets in human multiple myeloma cells

Abstract PU.1 is a hematopoietic transcription factor belonging to the Ets-family. It is identical to the Spi-1 oncogene, which is implicated in spleen focus-forming virus-induced murine erythroleukemias. PU.1 seems to be required for early development of multiple hematopoietic lineages, but its expression in mature cells is preferentially observed in cells of the B-cell-and monocyte/macrophage-differentiation lineage. It binds the so-called Pu box, an important tissue-specific regulatory DNA element present in a number of genes expressed in these cell lineages. We have analyzed the expression and activity of PU.1 during human B-cell development using a panel of B-cell lines representing different stages of maturation, from early precursors to differentiated plasma cells. PU.1 mRNA expression and PU.1 DNA binding activity, as measured by Northern blot analysis and electrophoretic mobility shift assay, respectively, were evident in cell lines representing pro-B, pre- B, and mature B cells. We could also show Pu box-dependent transactivation of a reporter gene in transient transfections in these cell lines. In contrast, in a number of multiple myeloma cell lines, representing differentiated, plasma cell-like B cells, PU.1 DNA binding activity, mRNA expression, and Pu box-dependent transactivation were absent or detectable at a very low level. In lymphoblastoid cell lines, which exemplify an intermediate stage of B-cell differentiation, a reduced expression and activity were observed. The findings in the human multiple myeloma cell lines represent the first examples of B cells with downregulated PU.1 expression and apparently contradict observations in the murine system in which PU.1 is expressed and active in plasmacytoma cell lines. At present, it is unclear whether the lack of PU.1 expression and activity in human multiple myeloma cell lines represents a malignancy-associated defect in these cells or exemplifies a normal developmental regulation in terminally differentiated B cells.

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NFκB Signaling and Mcl-1 Are Critical in B Cell Maturation Antigen-Promoted Multiple Myeloma Cell Growth and Survival

Blood ◽

10.1182/blood.v124.21.3384.3384 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3384-3384

Author(s):

Chirag Acharya ◽

Gang An ◽

Mike Y Zhong ◽

Michele Cea ◽

Antonia Cagnetta ◽

...

Keyword(s):

Multiple Myeloma ◽

Dna Binding ◽

B Cell ◽

Binding Activity ◽

Cell Maturation ◽

Dependent Manner ◽

Dna Binding Activity ◽

B Cell Maturation ◽

Mouse Xenograft Model ◽

B Cell Maturation Antigen

Abstract B cell maturation antigen (BCMA), selectively elevated in malignant plasma cells, is an ideal target antigen for immunotherapies for multiple myeloma (MM). Most recently, we reported novel antagonistic anti-BCMA antibody drug conjugates (ADCs) showing potent and specific anti-MM activities via effector cell-dependent and -independent mechanisms in vitro and in vivo (Blood 2014; 123:3128) We here further characterize molecular mechanisms of BCMA activation in MM cells in the bone marrow microenvironment by directly manipulating BCMA receptor levels in MM cells and ligation of a proliferation-inducing ligand (APRIL) to MM cells. Three MM cell lines H929, MM1S, and RPMI8226 with highest, medium, and low BCMA, respectively, were either transfected with lentiviruses of BCMA shRNA or cDNA. First, downregulation of BCMA significantly blocked viability of all 3 MM cells and induced caspase3/7 activities, which led to potent reduction of colony formation in a 3-week methylcellulose culture. Next, MM1R and H929 transfectants with the Doxycyclin (dox)-inducible lentiviral expression vector pTRIPZ shBCMA were generated. Time-dependent BCMA reduction only occurred in dox (1 ug/ml)-containing media. Dox-dependent BCMA inhibition was followed by decreased anti-apoptotic genes (Mcl1, Bcl-2, XIAP, NAIP, NFκB1, NFκB2) and proliferative genes (CCND2, CDK4/6, c-MYC). Conversely, overexpression of BCMA in RPMI8226 by either pCMV6/BCMA vector or pLocBCMA lentiviruses significantly increased NFκB (p65, p50, p52) DNA binding activity. Anti-apoptotic gene and cell proliferation genes were also up-regulated in BCMA-overexpressing MM cells. In addition, osteoclast activation factors MIP-1α/β, SDF-1, angiogenesis factors (VEGF, PECAM-1), adhesion proteins (CD44, ICAM1), as well as immunosuppressive factor TGFβ were augmented in BCMA-overexpressing MM cells. Importantly, opposite effects on these downstream genes were seen in BCMA-knockdown MM cells. Moreover, stimulation of 3 MM cells by APRIL robustly induces NFκB DNA binding activity (p65, p50, and p52, to a lesser extend) and activates PI3K/AKT and ERK1/2 signaling. APRIL also induces pro-survival/anti-apoptotic targets (BCL2A1, NFκB1, NFκB2) and chemotactic/osteoclast activating factors (MIP1α and MIP1β) in a dose-dependent manner. Angiogenesis and adhesion/chemoattractant factors (VEGF, IL-8, CXCL10, and RANTES) were also significantly induced upon APRIL stimulation. In contrast, BCMA-Fc protein that blocks APRIL binding to BCMA, inhibits secretion of these cytokines/chemokines, indicating specific response of engagement of BCMA by APRIL in BCMA-expressing MM cells. APRIL induced adhesion and migration of MM cells whereas BCMA-Fc blocked APRIL-induced responses. Finally, RPMI8226/pLocBCMA cells induce earlier tumor onset and more tumor growth in mouse xenograft model when compared with control RPMI8226 cells. In contrast, pTRIPZ shBCMA H929 cells induce significantly less tumor formation and further prolong survival of mice fed with dox(2 ug/ml)-containing water than those without dox. Together, these results define molecular regulators of active APRIL/BCMA signaling cascade in the MM BM milieu, further supporting targeting APRIL/BCMA in MM. Disclosures Anderson: Celgene: Consultancy; Sanofi-Aventis: Consultancy; Onyx: Consultancy; Acetylon: Scientific Founder, Scientific Founder Other; Oncoprep: Scientific Founder Other; Gilead Sciences: Consultancy.

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NF-kB as a Regulator of FA/BRCA Gene Expression in Multiple Myeloma.

Blood ◽

10.1182/blood.v110.11.3508.3508 ◽

2007 ◽

Vol 110 (11) ◽

pp. 3508-3508

Author(s):

Vasco A. Oliveira ◽

Linda Mathews ◽

Danielle Yarde ◽

Xingyu Wang ◽

David Boulware ◽

...

Keyword(s):

Gene Expression ◽

Multiple Myeloma ◽

Drug Resistance ◽

Dna Binding ◽

Cell Lines ◽

Myeloma Cell ◽

Binding Activity ◽

Acquired Drug Resistance ◽

Brca Gene ◽

Dna Binding Activity

Abstract Results to date argue compellingly that disruption of FA/BRCA gene expression plays a pivotal role in human somatic carcinogenesis. Melphalan, a DNA cross-linker, is one of the most widely used and effective drugs in the treatment of multiple myeloma (MM). Although most patients respond to standard and high dose melphalan, eventually patients acquire resistance and develop progressive disease. In 1991, our laboratory reported that acquired resistance in a human myeloma cell line was associated with reduced DNA crosslinks, elevated glutathione levels, and increased radiation survival (Cancer Res. 5:993; 1991). Most recently, we reported that the melphalan-resistant myeloma cell lines, 8226/LR5 and U266/LR6, showed a significant increase in several FA/BRCA genes compared to drug-sensitive cells, and that enhanced interstrand crosslink (ICL) repair via this signaling pathway contributes to acquired drug resistance in melphalan resistant cell lines (Blood 10:698; 2005). Here, we report that IKKa is constitutively phosphorylated in unstimulated 8226/LR5 cells, but not in melphalan-sensitive control cells. The specific phosphorylation of IKKa leads to an increase in basal NF-kB DNA binding activity, and 8226/LR5 cells are found to be markedly sensitive to BMS-345541 (a highly selective inhibitor of IkB) relative to control cells. Importantly, a cytotoxic dose of BMS-345541 induces a dramatic decrease in FA/BRCA gene expression, and a concomitant inhibition of NF-kB DNA binding activity in both 8226/S and 8226/LR5 cells. Furthermore, we show that 8226/LR5 cells experience the highest degree of direct binding between FANCD2 promoter and NF-kB/Rel family members, which, in turn, leads to an increase in basal FANCD2-specific NF-kB activity. Small-interfering RNA (siRNA)-mediated depletion of RelB and p50, but not other NF-kB subunits, in 8226 cells results in impaired NF-kB binding activity, and visible decrease in FANCD2 protein expression. Studies designed to dissect the role of NF-kB in acquired melphalan resistance are in progress, and the results will be presented. Our findings suggest that NF-kB functions as a regulator of FA/BRCA expression, and that this pathway represents a new target for preventing acquired drug resistance in myeloma patients.

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Gold Compound Auranofin (RIDAURA®) Induces Apoptosis of Human Myeloma Cells by Targeting STAT3 and NF-κB Pathways, with Clinical Potential.

Blood ◽

10.1182/blood.v114.22.3837.3837 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3837-3837 ◽

Cited By ~ 1

Author(s):

Morihiko Sagawa ◽

Aya Nakaya ◽

Tatsuki Tomikawa ◽

Yuichiro Haba ◽

Tomoe Nemoto ◽

...

Keyword(s):

Rheumatoid Arthritis ◽

Multiple Myeloma ◽

Drug Resistance ◽

Dna Binding ◽

Clinical Outcome ◽

Binding Activity ◽

Gold Compound ◽

Constitutive Activation ◽

Dna Binding Activity ◽

Induced Apoptosis

Abstract Abstract 3837 Poster Board III-773 Multiple myeloma (MM) is a plasma cell malignancy that remains incurable despite the use of conventional chemotherapy. Recent advances in the field of cell biology could lead to new therapeutic agents based upon molecular targeting, such as thalidomide and proteasome inhibitor bortezomib. These agents have remarkable activity against both newly diagnosed and refractory patients with MM, but prolonged exposure to them may result in the development of de novo drug resistance. Thus, it is necessary to identify and validate novel agents with less toxicity to overcome drug resistance and to improve the clinical outcome of MM. Progression and chemoresistance are thought to involve IL-6, whose expression is induced by NF-κB, through its regulation of the growth and survival of MM cells. IL-6 leads to constitutive activation of STAT3, which in turn results in the expression of high levels of Mcl-1. Thus, the constitutive activation of both NF-κB and STAT3 plays an important role in MM cell proliferation, and both molecules are important targets for the treatment of MM. Auranofin (AF: RIDAURA®; GSK) is a coordinated gold compound that has been widely used for the treatment of rheumatoid arthritis based on its anti-inflammatory properties through the inhibition of NF-κB activation. Therefore, assuming that AF has the potency to induce apoptosis in MM cells by interfering with NF-κB and STAT pathways, it may become a candidate for a novel therapeutic agent. To address our hypothesis, the effects of AF on inducing apoptosis of various MM cells were examined. Further, the molecular mechanism of AF-induced apoptosis in MM cells was investigated. AF inhibited the growth of U266 cells in a time- and dose-dependent manner with IC50 of 50 nM at 24 h. AF significantly induced cell cycle arrest at the G1 phase and subsequent apoptosis of U266 cells. AF-induced apoptosis in various human MM cell lines and CD138-positive plasma cells from patients with MM involved the activation of caspases-3, -8, and -9. Treatment with AF inhibited the constitutive and IL-6-induced activation of STAT3, and then downregulated the expression of Mcl-1 but not that of Bcl-2 or Bcl-xL proteins. To clarify the biological significance of Mcl-1 in AF-induced apoptosis of MM cells, Mcl-1 expression vector (pEGFP-hmcl-1) and control vector were introduced into U266 cells (designated as U266/mcl and U266/neo cells). Induction of apoptosis by AF was abrogated in U266/mcl, but not in U266/neo cells. We next examined the effect of AF on the DNA binding activity of STAT3. Electrophoretic mobility gel shift assay (EMSA) using U266 nuclear extracts demonstrated that IL-6-induced STAT3 binding activity was inhibited by the presence of AF. These results suggest that AF inhibits the IL-6-induced JAK/STAT pathway selectively and induces apoptosis in MM cells via the downregulation of Mcl-1. In addition, AF downregulated the activation of NF-κB in an IκB-independent manner, and also inhibited DNA binding activity in U266 cells. Although NF-κB inhibitory peptide (SN-50) did not directly induce the expression of Mcl-1, the combination of SN-50 with AF reduced the levels of Mcl-1, suggesting that inhibition of NF-κB potentiates the apoptotic effect of AF. Finally, our in vitro data prompted us to examine whether or not the effects of AF are equally valid in a clinical settings. The protocol for assessing the clinical benefit of oral administration of AF (RIDAURA®) in patients with relapsed or refractory MM was approved by the Ethics Committee of Saitama Medical University. Since 2008, 7 patients with refractory MM were treated with oral RIDAURA® 6 mg daily. The median follow-up for patients was 7.5 months. All 7 patients responded to AF, including one PR (partial response) and 6 SD (stable disease) with no progression. No adverse events have been observed to date. Details of the clinical outcome will be presented. In conclusion, gold compound AF inhibited constitutive activation of both STAT3 and NF-κB, resulting in the downregulation of anti-apoptotic Mcl-1 protein in MM cells with clinical relevance. A low pharmacological concentration (50 nM) of AF is widely employed for the treatment of rheumatoid arthritis without any side effects; therefore, it may be used to treat MM without the risk of severe toxicity. We propose that AF (RIDAURA®) may have potential use as a new molecular-targeted agent for the treatment of MM. Disclosures: Off Label Use: Auranofin in the treatment of multiple myeloma.

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The Mechanism of Constitutive NF-kB Activity in Myeloma Cell Lines.

Blood ◽

10.1182/blood.v110.11.4740.4740 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4740-4740

Author(s):

Jakia Amin ◽

Ken-ichiro Otsuyama ◽

Abul Islam ◽

Karim Shamsasenjan ◽

Shamim Mohd Iqbal ◽

...

Keyword(s):

Dna Binding ◽

Western Blot ◽

Cell Lines ◽

Target Gene ◽

Target Genes ◽

Constitutive Expression ◽

Myeloma Cell ◽

Binding Activity ◽

Myeloma Cells ◽

Dna Binding Activity

Abstract [Purpose] NF-kB has a key function in the transformation, proliferation and invasion of cancer cells as well as in resistance to radiotherapy and chemotherapy. Although constitutive NF-kB activation has been reported in many human tumors, the underlying factors and mechanisms responsible for constitutive NF-kB activation in myeloma cells has not been known. The purpose of this study was to determine the mechanism of constitutive NF-kB activity in myeloma cell lines and quantification of NF-kB activity in primary myeloma cells by expression of CD54 (a NF-kB target gene). [Methods] We checked the constitutive expression of NF-kB family proteins by western blot analysis and possible dimer formation of different NF-kB family members by Immunoprecipitation-western blot reaction. Constitutive NF-kB DNA-binding activity and dimers that are responsible for NF-kB activity were analyzed by electrophoretic mobility shift assay (EMSA). Moreover, expression of different NF-kB target genes was done by RT-PCR. [Results and discussion] Constitutive NF-kB activity was determined in myeloma cell lines and our results suggested that p50/RelB, p50/p50, p50/p52 dimers might be responsible for this. We also analyzed several NF-kB target gene expressions and found that the intensity of CD54 expression was positively correlated with total NF-kB DNA binding activity. Although there is no method to quantified NF-kB activity, it can be determine in terms of its target genes e.g. CD54. Therefore, this study provide the frame work for understanding the molecular mechanism of constitutive activation of NF-kB and would help to quantify NF-kB activation in primary myeloma cells.

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The hematopoietic transcription factor PU.1 is downregulated in human multiple myeloma cell lines

Blood ◽

10.1182/blood.v86.7.2747.bloodjournal8672747 ◽

1995 ◽

Vol 86 (7) ◽

pp. 2747-2753 ◽

Cited By ~ 2

Author(s):

M Pettersson ◽

C Sundstrom ◽

K Nilsson ◽

LG Larsson

Keyword(s):

Multiple Myeloma ◽

B Cells ◽

B Cell ◽

Cell Lines ◽

Myeloma Cell ◽

Binding Activity ◽

Multiple Myeloma Cell ◽

Dna Binding Activity ◽

Human Multiple Myeloma ◽

Expression And Activity

PU.1 is a hematopoietic transcription factor belonging to the Ets-family. It is identical to the Spi-1 oncogene, which is implicated in spleen focus-forming virus-induced murine erythroleukemias. PU.1 seems to be required for early development of multiple hematopoietic lineages, but its expression in mature cells is preferentially observed in cells of the B-cell-and monocyte/macrophage-differentiation lineage. It binds the so-called Pu box, an important tissue-specific regulatory DNA element present in a number of genes expressed in these cell lineages. We have analyzed the expression and activity of PU.1 during human B-cell development using a panel of B-cell lines representing different stages of maturation, from early precursors to differentiated plasma cells. PU.1 mRNA expression and PU.1 DNA binding activity, as measured by Northern blot analysis and electrophoretic mobility shift assay, respectively, were evident in cell lines representing pro-B, pre- B, and mature B cells. We could also show Pu box-dependent transactivation of a reporter gene in transient transfections in these cell lines. In contrast, in a number of multiple myeloma cell lines, representing differentiated, plasma cell-like B cells, PU.1 DNA binding activity, mRNA expression, and Pu box-dependent transactivation were absent or detectable at a very low level. In lymphoblastoid cell lines, which exemplify an intermediate stage of B-cell differentiation, a reduced expression and activity were observed. The findings in the human multiple myeloma cell lines represent the first examples of B cells with downregulated PU.1 expression and apparently contradict observations in the murine system in which PU.1 is expressed and active in plasmacytoma cell lines. At present, it is unclear whether the lack of PU.1 expression and activity in human multiple myeloma cell lines represents a malignancy-associated defect in these cells or exemplifies a normal developmental regulation in terminally differentiated B cells.

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