High Expression of NEK2 Mediated By p53 Contributes to Progression and Relapse of Multiple Myeloma

Abstract High expression of NEK2 mediated by p53 contributes to progression and relapse of multiple myeloma Xiangling Feng1,2, Jiaojiao Guo1, Bowen Ouyang2, Yinghong Zhu1,Gang An3, Hao Zhen1, Jiliang Xia1, Yongjun Guan1, Xinying Zhao2, Lugui Qiu3, Jiaxi Zhou3, Fenghuang Zhan4,Wen Zhou1 1, Cancer Research Institute,Central South University, Changsha 410078, China. 2Xiang Ya School of Public Health, Central South University, Changsha, Hunan, China. USA. 3State Key Laboratory of Experimental Hematology, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Science & Peking Union Medical College, Tianjin, China. 4Department of Internal Medicine, Division of Hematology, Oncology, and Blood & Marrow Transplantation, University of Iowa, Iowa City, USA. E-mail: [email protected]. Background: Loss of p53 is an independent prognostic factor in patients with multiple myeloma (MM). Our previous studies found abnormal high expression of NEK2 was closely related to the poor prognosis and drug resistance of myeloma patients. However, it's unclear how NEK2 was up-regulated in MM. Through bioinformatics analysis, the binding site of p53 protein is found in NEK2 promoter, but the relationship and function of p53 and NEK2 in MM are poorly understood. Materials and Methods: In this study, p53-/- MM cell lines (ARP1 and KMS11) and p53 p53+/+ MM cell lines (MM1S and H929) were used for investigating the role of NEK2 in p53-/- MM cell. FISH was performed on interphase nuclei of MM primary cells to detect p53 and NEK2 copy numbers. Chromatin immunoprecipitation and fluorescence reporter system were applied for examining the binding site of p53 protein in the distal NEK2 promoter. CGH and RNA-seq were performed to validate copy number changes and variations in the expression of several transcripts. Results: The top 10% of MM patients with the highest NEK2 expression and lowest p53 had a significantly inferior OS (P<0.001) in TT2 and TT3 patients (GSE2658) and the expression of NEK2 increased significantly in myeloma cells during chemotherapy(GSE19554), while p53 decreased with the disease progression, suggesting a strong relationship with drug resistance. Single cell PCR showed increased NEK2 expression correlated with decreased p53 expression in single CD138+ plasma cell. FISH confirmed the loss of p53 in CD138+ plasma cells with amplification of NEK2 copies. Furthermore, NEK2 was also high expressed in p53 low expressed MM cells by Immunofluorescence (IF) (P<0.01). In addition, NEK2 was upregulated in p53-/- MM cell lines and HEK293 cells by deleted p53 gene with CRISPR technique both on mRNA and protein level (P<0.01), suggesting a negative correlation between the p53 and the expression level of NEK2. Meanwhile, when p53 deletion and NEK2 overexpression occur simultaneously, the phenomena of asymmetric mitosis and multipolar division are more obvious (P<0.001), suggested that the double hit of p53 deletion and NEK2 overexpression increases the chromosomal instability. Further in vivo study indicated the subcutaneous tumorigenesis in p53 deletion and NEK2 overexpression group was significantly greater than that of the single overexpression of NEK2 and deletion of p53 group (P<0.001), suggested that NEK2 overexpression and p53 deletion enhances the tumorigenic ability in vivo. While down-regulation of NEK2 by shRNA in p53 deletion cells, cell growth was inhibited in vitro and in vivo.To explore the relationship between p53 and NEK2, chromatin immunoprecipitation and fluorescence reporter system showed that p53 could bind to the promoter region of NEK2 and regulate its transcriptional expression. Further CGH analysis of the deletion of p53 expression in HEK293 cells can cause 1q21.4-44 amplification of the chromosome region of the NEK2 directly, which further confirmed by FISH. Finally, RNA-seq revealed several chromosome instability genes were abnormal expressed in NEK2 overexpression and p53 deletion double-hit group. Conclusion: In summary, p53 deletion and NEK2 overexpression induced cancer cell drug resistance, proliferation and chromosomal instability. p53 could bind to the promoter region of NEK2 and cause NEK2 amplification. Down-regulation of NEK2 by shRNA in p53 deletion cells inhibited cell growth in vitro and in vivo. Thus,The significance of this study will provide the pre-clinical application of the NEK2 inhibitor to overcome the drug resistance induced by p53 in MM. Disclosures No relevant conflicts of interest to declare.

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TAS-117, a Novel Selective Akt Inhibitor Demonstrates Significant Growth Inhibition in Multiple Myeloma Cells in Vitro and in Vivo

Blood ◽

10.1182/blood.v120.21.942.942 ◽

2012 ◽

Vol 120 (21) ◽

pp. 942-942 ◽

Cited By ~ 1

Author(s):

Naoya Mimura ◽

Hiroto Ohguchi ◽

Diana Cirstea ◽

Francesca Cottini ◽

Gullu Topal Gorgun ◽

...

Keyword(s):

Multiple Myeloma ◽

Drug Resistance ◽

Cell Growth ◽

Board Of Directors ◽

Cell Lines ◽

Akt Inhibitor ◽

Advisory Committees ◽

Significant Growth Inhibition

Abstract Abstract 942 The PI3K/Akt pathway mediates multiple myeloma (MM) cell growth and drug resistance, and targeting this molecule is a promising therapeutic option. In this study, we examined anti-MM activities of TAS-117 (TAIHO PHARMACEUTICAL CO., LTD., JAPAN), a selective potent Akt inhibitor in MM cell lines including MM.1S, MM.1R, OPM1 and H929 cells with high level of baseline Akt phosphorylation. TAS-117 induced significant growth inhibition in these cell lines, associated with downregulation of phosphorylation (Ser473 and Thr308) of Akt and downstream molecule FKHR/FKHRL1, without cytotoxicity in normal peripheral blood mononuclear cells. TAS-117 triggered G0/G1 arrest followed by apoptosis, evidenced by increased annexin V-positive cells, in both MM.1S and H929 cell lines. Apoptosis was further confirmed by cleavage of caspase-8, -3 and PARP. Interestingly, TAS-117 also induced: autophagy, evidenced by increased LC3-II; as well as endoplasmic reticulum (ER) stress, confirmed by induction of phospho-eIF2α, phospho-IRE1α and a molecular chaperone BiP/GRP78. Since the bone marrow (BM) microenvironment plays a crucial role in MM cell pathogenesis including drug resistance, we further examined the effect of TAS-117 in the presence of BM stromal cells (BMSCs). TAS-117 induced significant cytotoxicity in MM cells even in the presence of BMSCs, associated with downregulation of phospho-Akt. Importantly, TAS-117 inhibited secretion of IL-6 from BMSCs, and exogenous IL-6 and IGF-1 did not block cytotoxicity induced by this agent. We have previously shown the bortezomib activates Akt, and that Akt inhibition with bortezomib triggers synergistic MM cell cytotoxicity. TAS-117 enhanced bortezomib-induced cytotoxicity in MM.1S cells, associated with increased CHOP followed by PARP cleavage, suggesting that TAS-117 augments bortezomib-induced ER stress and apoptotic signaling. TAS-117 also enhanced cytotoxicity induced by other therapeutic agents (ie, rapamycin, dexamethasone, 17-AAG) in MM.1S cells. Finally, we examined anti-MM activities of TAS-117 in a xenograft murine model. Oral administration of TAS-117 for 14 days significantly inhibited growth of H929 plasmacytoma and was well tolerated. Taken together, the novel and selective Akt inhibitor TAS-117 blocks MM cell growth in vitro and in vivo, providing the preclinical framework for clinical evaluation of this agent to improve patient outcome in MM. Disclosures: Shimomura: TAIHO PHARMACEUTICAL CO., LTD.: Employment. Utsugi:TAIHO PHARMACEUTICAL CO., LTD.: Membership on an entity's Board of Directors or advisory committees. Anderson:Celgene, Millennium, BMS, Onyx: Membership on an entity's Board of Directors or advisory committees; Acetylon, Oncopep: Scientific Founder, Scientific Founder Other.

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

Blood ◽

10.1182/blood-2018-99-113765 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1945-1945

Author(s):

Kylee H Maclachlan ◽

Andrew Cuddihy ◽

Nadine Hein ◽

Carleen Cullinane ◽

Simon J. Harrison ◽

...

Keyword(s):

Cell Cycle ◽

Multiple Myeloma ◽

Drug Resistance ◽

Cell Lines ◽

Cellular Response ◽

Single Agent ◽

Resistant Cell ◽

Rdna Transcription

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

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P53 Pathway Activation Mediated High c-MAF Expression Is Associated with Overall and Post-Progression Survival in Multiple Myeloma

Blood ◽

10.1182/blood-2019-125172 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4330-4330

Author(s):

Eiko Yamane ◽

Tsukasa Oda ◽

Masanobu Sunaga ◽

Yuki Murakami ◽

Rei Ishihara ◽

...

Keyword(s):

Multiple Myeloma ◽

Drug Resistance ◽

Cell Lines ◽

Poor Prognosis ◽

High Expression ◽

Cyclin D2 ◽

P53 Overexpression ◽

P53 Pathway ◽

And Control

Background: Chromosomal abnormalities are strongly associated with prognosis of multiple myeloma (MM). Among them, t (14;16) resulting in high expression of c-MAF, and t (14;20) resulting in high expression of MAFB, lead to poor prognosis. However, clinical significance and mechanisms underlying high c-MAF and MAFB expression without these translocations have not been fully elucidated. Methods: A total of 96 MM and 38 MGUS patients, 10 controls, and 9 MM cell lines were included in this study. RNA was extracted from purified CD138+ plasma cells from bone marrow (BM) mononuclear cells. c-MAF, MAFB, p53, and p21 RNA expressions were determined by RQ-PCR. Their expression levels were normalized against ACTB levels and calculated with 2-ΔΔCt value. Inhibition studies using BRD4 inhibitor JQ1, and MYC inhibitor10058-F4, and MM cell lines expressing doxycycline-inducible p53 (Tet-on p53) or c-MAF knockdown (KD) with siRNA were used for in vitro studies. Results: c-MAF expression in MM patients was higher than in MGUS patients and control (median level 0.043, 0.025, 0.002, p<0.001), but MAFB expression did not differ between MM, MGUS, and control (p=0.371). Although c-MAF expression level was significantly higher in MM with t (14;16) (p=0.0018), high c-MAF expression was observed in patients without the translocations, suggesting a role of c-MAF in MM progression. Other cytogenetic abnormalities such as del 17p, or t (4;14), did not affect c-MAF expression. Overall survival (OS) of MM patients with high c-MAF expression was significantly inferior compared to the patients with low c-MAF expression (HR 2.46, p=0.002, 2 years survival rate 42.9% vs. 72.7%, median 20.3 months vs. not reached), but progression-free survival (PFS) did not differ (p=0.551). Instead, post-progression survival (PPS) was significantly shorter in the patients with high c-MAF expression (HR 4.67, p<0.001, two years survival 0% vs. 60.2%, median 3.6 months vs. 49.3 months) suggesting that c-MAF confers drug resistance to residual disease. MAFB expression did not affect OS, PFS, and PPS. Reduction of cyclin D2 expression by c-MAF KD in vitro and a positive correlation between c-MAF and cyclin D2 expression (r=0.29, p<0.001) in vivo in patients, support previous reports describing that MM proliferation is induced by c-MAF via cyclin D2. c-MAF was not correlated with APOBEC3B expression. c-MAF expression was positively correlated with MDM2 and MYC (r=0.387, p=0.03 and r=0.221, p=0.019, respectively) but not with p53 and PUMA. A tendency of positive correlation was also observed with p21/CDK1NA (r=0.304, p=0.085). p53 overexpression from the Tet-on p53 and p53 stabilization by nutlin-3 increased c-MAF expression in three cell lines with t (14;16). c-MAF KD together with p53 overexpression significantly suppressed the proliferation, implying synthetic lethality. These findings suggest that c-MAF expression is upregulated in response to proliferation arrest induced by the p53 pathway; suppressing this responsive c-MAF expression abrogates cell growth more efficiently. JQ1 and CPI203 increased c-MAF expression in cell lines with t (14;16), suggesting that c-MAF expression is not controlled by super-enhancers. Conclusion: Although the mechanism of high expression of c-MAF and MAFB has previously been reported in IgH translocation, the relationship between c-MAF and drug resistance remains to be determined. The induction of c-MAF expression by tumor suppressor gene p53 is suggested to be a mechanism underlying poor prognosis of MM with t (14;16), and suppressing c-MAF expression with chemotherapeutic drugs might prevent emergence of drug resistance in residual tumor cells. Disclosures Handa: Ono: Research Funding.

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Targeting NSD2-mediated SRC-3 liquid–liquid phase separation sensitizes bortezomib treatment in multiple myeloma

Nature Communications ◽

10.1038/s41467-021-21386-y ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Jing Liu ◽

Ying Xie ◽

Jing Guo ◽

Xin Li ◽

Jingjing Wang ◽

...

Keyword(s):

Multiple Myeloma ◽

Drug Resistance ◽

Phase Separation ◽

Liquid Phase ◽

Liquid Phase Separation ◽

Acquired Drug Resistance ◽

Bortezomib Treatment ◽

Liquid Liquid Phase Separation

AbstractDevelopment of chemoresistance is the main reason for failure of clinical management of multiple myeloma (MM), but the genetic and epigenetic aberrations that interact to confer such chemoresistance remains unknown. In the present study, we find that high steroid receptor coactivator-3 (SRC-3) expression is correlated with relapse/refractory and poor outcomes in MM patients treated with bortezomib (BTZ)-based regimens. Furthermore, in immortalized cell lines, high SRC-3 enhances resistance to proteasome inhibitor (PI)-induced apoptosis. Overexpressed histone methyltransferase NSD2 in patients bearing a t(4;14) translocation or in BTZ-resistant MM cells coordinates elevated SRC-3 by enhancing its liquid–liquid phase separation to supranormally modify histone H3 lysine 36 dimethylation (H3K36me2) modifications on promoters of anti-apoptotic genes. Targeting SRC-3 or interference of its interactions with NSD2 using a newly developed inhibitor, SI-2, sensitizes BTZ treatment and overcomes drug resistance both in vitro and in vivo. Taken together, our findings elucidate a previously unrecognized orchestration of SRC-3 and NSD2 in acquired drug resistance of MM and suggest that SI-2 may be efficacious for overcoming drug resistance in MM patients.

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PIWIL1 Drives Chemoresistance in Multiple Myeloma by Modulating Mitophagy and the Myeloma Stem Cell Population

Frontiers in Oncology ◽

10.3389/fonc.2021.783583 ◽

2022 ◽

Vol 11 ◽

Author(s):

Yajun Wang ◽

Lan Yao ◽

Yao Teng ◽

Hua Yin ◽

Qiuling Wu

Keyword(s):

Multiple Myeloma ◽

Drug Resistance ◽

Stem Cell ◽

Cell Population ◽

Side Population ◽

Chemotherapeutic Agents ◽

Stem Cell Population ◽

Exact Function

As an important member of the Argonaute protein family, PIWI-like protein 1 (PIWIL1) plays a key role in tumor cell viability. However, the exact function of PIWIL1 in multiple myeloma (MM) and the underlying mechanism remain unclear. Here, we revealed that PIWIL1 was highly expressed in myeloma cell lines and newly diagnosed MM patients, and that its expression was notably higher in refractory/relapsed MM patients. PIWIL1 promoted the proliferation of MM cells and conferred resistance to chemotherapeutic agents both in vitro and in vivo. More importantly, PIWIL1 enhanced the formation of autophagosomes, especially mitophagosomes, by disrupting mitochondrial calcium signaling and modulating mitophagy-related canonical PINK1/Parkin pathway protein components. Mitophagy/autophagy inhibitors overcome PIWIL1-induced chemoresistance. In addition, PIWIL1 overexpression increased the proportion of side population (SP) cells and upregulated the expression of the stem cell-associated genes Nanog, OCT4, and SOX2, while its inhibition resulted in opposite effects. Taken together, our findings demonstrated that PIWIL1 induced drug resistance by activating mitophagy and regulating the MM stem cell population. PIWIL1 depletion significantly overcame drug resistance and could be used as a novel therapeutic target for reversing resistance in MM patients.

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Deacetylation Induced Nuclear Condensation of HP1γ Promotes Multiple Myeloma Drug Resistance

10.21203/rs.3.rs-698350/v1 ◽

2021 ◽

Author(s):

Zhiqiang Liu ◽

Xin Li ◽

Sheng Wang ◽

Ying Xie ◽

Hongmei Jiang ◽

...

Keyword(s):

Multiple Myeloma ◽

Dna Damage ◽

Drug Resistance ◽

Heterochromatin Protein 1 ◽

Nuclear Condensation ◽

Repair Capacity ◽

Acquired Drug Resistance ◽

Histone Deacetylase 1

Abstract Acquired chemoresistance to proteasome inhibitors (PIs) is a major obstacle that results in failure to manage patients with multiple myeloma (MM) in the clinic; however, the key regulators and underlying mechanisms are still unclear. In this study, we found that high levels of a chromosomal modifier, heterochromatin protein 1 gamma (HP1γ), are accompanied by a low acetylation level in bortezomib-resistant (BR) MM cells, and aberrant DNA repair capacity is correlated with HP1γ overexpression. Mechanistically, the deacetylation of HP1γ at lysine 5 by histone deacetylase 1 (HDAC1) alleviates HP1γ ubiquitination, and the stabilized HP1γ recruits the mediator of DNA damage checkpoint 1 (MDC1) to induce DNA damage repair. Simultaneously, deacetylation modification and MDC1 recruitment enhance the nuclear condensate of HP1γ, which facilitates the chromatin accessibility of genes governing sensitivity to PIs, such as FOS, JUN and CD40. Thus, targeting HP1γ stability using the HDAC1/2 inhibitor, romidepsin, sensitizes PIs treatment and overcomes drug resistance both in vitro and in vivo. Our findings elucidate a previously unrecognized role of HP1γ in the acquired drug resistance of MM and suggest that targeting HP1γ may be efficacious for overcoming drug resistance in MM patients.

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Irinotecan Resistance – In Search of New Markers in CRC.

10.21203/rs.3.rs-1088696/v1 ◽

2021 ◽

Author(s):

Jakub Kryczka ◽

Joanna Boncela

Keyword(s):

Drug Resistance ◽

Cell Lines ◽

Expression Profiles ◽

Resistance Mechanism ◽

Interaction Network ◽

In Vitro Study ◽

Cancer Drug ◽

New Genes

Abstract Colorectal cancer (CRC) is one of the most prominent causes of cancer death worldwide. Chemotherapeutic regimens consisting of different drugs combinations such as 5-fluorouracil, and oxaliplatin (FOLFOX) or irinotecan (FOLFIRI) have been proven successful in the treatment of CRC. However, chemotherapy often leads to the acquisition of cancer drug resistance followed by metastasis and in the aftermath therapeutic failure. The molecular mechanism responsible for drug resistance is still unclear. The systemic search for new biomarkers of this phenomenon may identify new genes and pathways. To understand the drug resistance mechanism in CRC, the in vitro study based on the molecular analysis of drug-sensitive cells lines vs drug-resistant cells lines has been used. In our study to bridge the gap between in vitro and in vivo study, we compared the expression profiles of cell lines and patient samples from the publicly available database to select the new candidate genes for irinotecan resistance. Using The Gene Expression Omnibus (GEO) database of CRC cell lines (HT29, HTC116, LoVo, and their respective irinotecan-resistant variants) and patient samples (GSE42387, GSE62080, and GSE18105) we compared the changes in the mRNA expression profile of the main genes involved in irinotecan body’s processing, such as transport out of the cells and metabolism. Furthermore, using a protein-protein interaction network of differently expressed genes between FOLFIRI resistant and sensitive CRC patients, we have selected top networking proteins (upregulated: NDUFA2, SDHD, LSM5, DCAF4, and COX10, downregulated: RBM8A, TIMP1, QKI, TGOLN2, and PTGS2). Our analysis provided several potential irinotecan resistance markers, previously not described as such.

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Antitumorigenic Effects of Inhibiting Ephrin Receptor Kinase Signaling by GLPG1790 against Colorectal Cancer Cell Lines In Vitro and In Vivo

Journal of Oncology ◽

10.1155/2020/9342732 ◽

2020 ◽

Vol 2020 ◽

pp. 1-16 ◽

Cited By ~ 1

Author(s):

Alessandro Colapietro ◽

Giovanni Luca Gravina ◽

Francesco Petragnano ◽

Irene Fasciani ◽

Bianca Maria Scicchitano ◽

...

Keyword(s):

Colorectal Cancer ◽

Cell Lines ◽

Receptor Expression ◽

Mutant Form ◽

Kinase Signaling ◽

P53 Expression ◽

Mesenchymal Transition ◽

Hct116 Cells

Erythropoietin-producing hepatocellular receptors (Eph) promote the onset and sustain the progression of cancers such as colorectal cancer (CRC), in which the A2 subtype of Eph receptor expression has been shown to correlate with a poor prognosis and has been identified as a promising therapeutic target. Herein, we investigated, in vitro and in vivo, the effects of treatment with GLPG1790, a potent pan-Eph inhibitor. The small molecule has selective activity against the EphA2 isoform in human HCT116 and HCT15 CRC cell lines expressing a constitutively active form of RAS concurrently with a wild-type or mutant form of p53, respectively. GLPG1790 reduced EPHA2 phosphorylation/activation and induced G1/S cell-cycle growth arrest by downregulating the expression of cyclin E and PCNA, while upregulating p21Waf1/Cip1 and p27Cip/Kip. The inhibition of ephrin signaling induced quiescence in HCT15 and senescence in HCT116 cells. While investigating the role of CRC-related, pro-oncogenic p53 and RAS pathways, we found that GLPG1790 upregulated p53 expression and that silencing p53 or inhibiting RAS (human rat sarcoma)/ERKs (extracellular signal-regulated kinase) signaling restrained the ability of GLPG1790 to induce senescence in HCT116 cells. On the other hand, HCT15 silencing of p53 predisposed cells to GLPG1790-induced senescence, whilst no effects of ERK inhibition were observed. Finally, GLPG1790 hindered the epithelial-mesenchymal transition, reduced the migratory capacities of CRC, and affected tumor formation in xenograft models in vivo more efficiently using HCT116 than HCT15 for xenografts. Taken together, our data suggest the therapeutic potential of GLPG1790 as a signal transduction-based therapeutic strategy in to treat CRC.

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JS-K, a GST-Activated Nitric Oxide Generator, Induces Apoptosis and Overcomes In Vitro Drug Resistance in Multiple Myeloma Cells.

Blood ◽

10.1182/blood.v106.11.1593.1593 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1593-1593

Author(s):

Tanyel Kiziltepe ◽

Kenji Ishitsuka ◽

Teru Hideshima ◽

Noopur Raje ◽

Norihiko Shiraishi ◽

...

Keyword(s):

Nitric Oxide ◽

Multiple Myeloma ◽

Drug Resistance ◽

Tumor Cells ◽

Cell Lines ◽

Biological Effects ◽

Treatment Modalities ◽

Cancer Drug ◽

Induced Apoptosis

Abstract Multiple myeloma (MM) is currently an incurable hematological malignancy. A major reason for the failure of currently existing therapies is the chemotherapeutic resistance acquired by the MM cells upon treatment. Overexpression of glutathione S-transferases (GST) has been shown as one possible mechanism of anti-cancer drug resistance in a broad spectrum of tumor cells. JS-K (O2-(2,4-Dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate) belongs to a class of pro-drugs which are designed to release nitric oxide (NO) on reaction with GST. JS-K can possibly turn GST overexpression to the tumor’s disadvantage by (1) consuming intracellular GSH and preventing drug inactivation; and (2) by exposing tumor cells to high intracellular concentrations of NO. JS-K has potent in vitro and in vivo anti-leukemic activity. The purpose of the present study is to examine the biological effects of JS-K on human MM cells. We demonstrate that JS-K has significant in vitro cytotoxicity on MM cell lines, with an IC50 of 0.3-2 mM at 48 hours. JS-K also induces cytotoxicity on cell lines that are resistant to conventional chemotherapy (i.e., MM1R, RPMI-Dox40, RPMI-LR5, RPMI-MR20). Importantly, no cytotoxic effects of JS-K were detected on peripheral blood mononuclear cells (PBMNC) obtained from healthy volunteers at these doses. Moreover, JS-K could overcome the survival and growth advantages conferred by interleukin-6 (IL-6) and insulin-like growth factor-1 (IGF-1), or by adherence of MM cells to bone marrow stromal cells (BMSC). JS-K caused a transient G2/M arrest followed by apoptosis, as determined by flow cytometric analysis using PI, Annexin V and Apo2.7 staining. JS-K-induced apoptosis was associated with caspase 8, 7, 9 and 3 activation. Interestingly, Fas was upregulated by JS-K, suggesting the involvement of death receptor pathway in induction of apoptosis. JS-K also triggered Mcl-1 cleavage and Bcl-2 phosphorylation, suggesting the involvement of mitochondrial pathway. In addition, apoptosis inducing factor (AIF), endonuclease G (EndoG) and cytochrome c were released into the cytosol during apoptosis. Taken together, these findings suggest the involvement of both intrinsic and extrinsic apoptotic pathways in JS-K-induced apoptosis in MM cells. In summary, our studies demonstrate that JS-K induces apoptosis and overcomes in vitro drug resistance in MM cells. Therefore, JS-K is a novel compound which carries significant potential to be included in the repertoire of existing treatment modalities for MM. Ongoing studies are delineating the mechanism of action of JS-K to provide the preclinical rationale for combination therapies to overcome drug resistance and improve patient outcome.

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CHIR258, a Novel Multi-Targeted Tyrosine Kinase Inhibitor, for the Treatment of t(4;14) Multiple Myeloma.

Blood ◽

10.1182/blood.v104.11.641.641 ◽

2004 ◽

Vol 104 (11) ◽

pp. 641-641 ◽

Cited By ~ 1

Author(s):

Suzanne Trudel ◽

Zhi Hua Li ◽

Ellen Wei ◽

Marion Wiesmann ◽

Katherine Rendahl ◽

...

Keyword(s):

Multiple Myeloma ◽

Mouse Model ◽

Tyrosine Kinase ◽

Cell Lines ◽

Small Molecule ◽

Kinase Inhibitor ◽

Wild Type ◽

Myeloma Cells

Abstract The t(4;14) translocation that occurs uniquely in a subset (15%) of multiple myeloma (MM) patients results in the ectopic expression of the receptor tyrosine kinase, Fibroblast Growth Factor Receptor3 (FGFR3). Wild-type FGFR3 induces proliferative signals in myeloma cells and appears to be weakly transforming in a hematopoeitic mouse model. The subsequent acquisition of FGFR3 activating mutations in some MM is associated with disease progression and is strongly transforming in several experimental models. The clinical impact of t(4;14) translocations has been demonstrated in several retrospective studies each reporting a marked reduction in overall survival. We have previously shown that inhibition of activated FGFR3 causes morphologic differentiation followed by apoptosis of FGFR3 expressing MM cell lines, validating activated FGFR3 as a therapeutic target in t(4;14) MM and encouraging the clinical development of FGFR3 inhibitors for the treatment of these poor-prognosis patients. CHIR258 is a small molecule kinase inhibitor that targets Class III–V RTKs and inhibits FGFR3 with an IC50 of 5 nM in an in vitro kinase assay. Potent anti-tumor and anti-angiogenic activity has been demonstrated in vitro and in vivo. We employed the IL-6 dependent cell line, B9 that has been engineered to express wild-type FGFR3 or active mutants of FGFR3 (Y373C, K650E, G384D and 807C), to screen CHIR258 for activity against FGFR3. CHIR258 differentially inhibited FGF-mediated growth of B9 expressing wild-type and mutant receptors found in MM, with an IC50 of 25 nM and 80 nM respectively as determined by MTT proliferation assay. Growth of these cells could be rescued by IL-6 demonstrating selectivity of CHIR258 for FGFR3. We then confirmed the activity of CHIR258 against FGFR3 expressing myeloma cells. CHIR258 inhibited the viability of FGFR3 expressing KMS11 (Y373C), KMS18 (G384D) and OPM-2 (K650E) cell lines with an IC50 of 100 nM, 250 nM and 80 nM, respectively. Importantly, inhibition with CHIR258 was still observed in the presence of IL-6, a potent growth factors for MM cells. U266 cells, which lack FGFR3 expression, displayed minimal growth inhibition demonstrating that at effective concentrations, CHIR258 exhibits minimal nonspecific cytotoxicity on MM cells. Further characterization of this finding demonstrated that inhibition of cell growth corresponded to G0/G1 cell cycle arrest and dose-dependent inhibition of downstream ERK phosphorylation. In responsive cell lines, CHIR258 induced apoptosis via caspase 3. In vitro combination analysis of CHIR258 and dexamethasone applied simultaneously to KMS11 cells indicated a synergistic interaction. In vivo studies demonstrated that CHIR258 induced tumor regression and inhibited growth of FGFR3 tumors in a plasmacytoma xenograft mouse model. Finally, CHIR258 produced cytotoxic responses in 4/5 primary myeloma samples derived from patients harboring a t(4;14) translocation. These data indicate that the small molecule inhibitor, CHIR258 potently inhibits FGFR3 and has activity against human MM cells setting the stage for a Phase I clinical trial of this compound in t(4;14) myeloma.

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