Discovery and Characterization of Chemical Inhibitors of UBC13.

Abstract Abstract 2950 Poly-ubiquitination of signaling proteins via lysine 63 (K63)-linked chains is recognized as a critical post-translational modification involved in activation of NF-kB and stress kinases in the context of signaling by Tumor Necrosis Factor Receptors (TNFRs), Toll-like receptors (TLRs), and antigen receptors. UBC13 is a K63-specific ubiquitin conjugating enzyme that partners with TNFR-associated factors (TRAFs) to mediate K63-linked ubiquitination. Gene ablation studies have shown UBC13 is required for NF-kB signaling induced by a variety of stimuli in specific types of immune cells, making it a potential target for certain cancers, autoimmune and inflammatory diseases. UBC13 operates together with obligatory cofactors, either UEV1A in the cytosol or MMS2 in the nucleus. The nuclear function of UBC13 is evolutionarily conserved, where it plays a critical role in double strand DNA repair, making UBC13 a potential chemo- and radio-sensitizer target for oncology. To identify chemical inhibitors of UBC13, we developed a HTS assay measuring UBC13-UEV1A enzymatic activity by TR-FRET, screening altogether ∼450,000 diverse compounds. Hit compounds were characterized using a rigorous testing funnel consisting of (a) informatics filtering against a database of > 100 HTS campaigns conducted with the same libraries, to eliminate promiscuous compounds; (b) counter-screens against E1, another cysteine-dependent enzyme (caspase-3), and against an irrelevant target formatted as a TR-FRET assay; and (c) ordering compounds from fresh powders and demonstrating reproducible concentration-dependent inhibition of UBC13. The surviving hits were then analyzed by cell-based assays for suppression of TRAF6 ubiquitination but not Mdm2-mediated ubiquitination of p53, resulting in 14 promising hits that included two chemical series. While suppressing TRAF6 ubiquitination (UBC13-dependent) in cells, these compounds did not interfere with either SUMOylation (UBC9-dependent) or NEDDylation (UBC12-dependent) of cellular proteins. UBC13 inhibitors also suppressed NF-kB activity (measured using stably integrated NF-kB-driven luciferase reporter gene) induced by PKC activators (Carma/Bcl-10/MALT pathway) and DNA damaging agent (Doxorubicin) but not by TNF-a. Investigations of the bioactivity of these UBC13 inhibitory compounds and their analogs will be described for a variety of hematolymphoid malignancies. (Supported by NIH R03-MH085677, NIH U54–005033, and by a fellowship grant from International Myeloma Foundation). Disclosures: No relevant conflicts of interest to declare.

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My D88-Dependent Interplay Between Myeloid and Endothelial Cells in the Initiation and Progression of Obesity-Associated Inflammatory Diseases

Blood ◽

10.1182/blood.v128.22.sci-44.sci-44 ◽

2016 ◽

Vol 128 (22) ◽

pp. SCI-44-SCI-44

Author(s):

Xiaoxia Li

Keyword(s):

Insulin Resistance ◽

Endothelial Cells ◽

Adipose Tissue ◽

Systemic Inflammation ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Inflammatory Diseases ◽

Critical Role ◽

Low Grade

Abstract Low-grade systemic inflammation is often associated with metabolic syndrome, which plays a critical role in the development of the obesity-associated inflammatory diseases, including insulin resistance and atherosclerosis. Here, we investigate how Toll-like receptor-MyD88 signaling in myeloid and endothelial cells coordinately participates in the initiation and progression of high fat diet-induced systemic inflammation and metabolic inflammatory diseases. MyD88 deficiency in myeloid cells inhibits macrophage recruitment to adipose tissue and their switch to an M1-like phenotype. This is accompanied by substantially reduced diet-induced systemic inflammation, insulin resistance, and atherosclerosis. MyD88 deficiency in endothelial cells results in a moderate reduction in diet-induced adipose macrophage infiltration and M1 polarization, selective insulin sensitivity in adipose tissue, and amelioration of spontaneous atherosclerosis. Both in vivo and ex vivo studies suggest that MyD88-dependent GM-CSF production from the endothelial cells might play a critical role in the initiation of obesity-associated inflammation and development of atherosclerosis by priming the monocytes in the adipose and arterial tissues to differentiate into M1-like inflammatory macrophages. Collectively, these results implicate a critical MyD88-dependent interplay between myeloid and endothelial cells in the initiation and progression of obesity-associated inflammatory diseases. Disclosures No relevant conflicts of interest to declare.

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A distal activation domain is critical in the regulation of the rat androgen receptor gene promoter

Biochemical Journal ◽

10.1042/bj2940779 ◽

1993 ◽

Vol 294 (3) ◽

pp. 779-784 ◽

Cited By ~ 19

Author(s):

C S Song ◽

S Her ◽

M Slomczynska ◽

S J Choi ◽

M H Jung ◽

...

Keyword(s):

Androgen Receptor ◽

Nucleotide Sequence ◽

Receptor Gene ◽

Critical Role ◽

Androgen Receptor Gene ◽

Luciferase Reporter ◽

Upstream Region ◽

Luciferase Reporter Gene ◽

Mobility Shift ◽

Cis Element

The far upstream region of the rat androgen receptor (AR) gene has been cloned, and the nucleotide sequence up to -2656 bp established. Nested deletion mutants of rat AR 5′ flanking sequences were ligated to the luciferase reporter gene, and their promoter activities were examined in transfected COS1 cells. Results show a critical cis-acting domain located between positions -960 and -940. Deletion of this cis element resulted in a greater than 90% decrease in the promoter activity. A nuclear protein that specifically binds to this 21-nucleotide sequence was identified by gel mobility shift analysis. The -960/-940 cis element has no identify to the binding sequence of any known transcription factor. Furthermore, the cognate binding protein is present in both rat and human (HeLa) cell nuclear extracts. We conclude that a novel trans-activator interacting at the -960/-940 region plays a critical role in the regulation of AR gene expression.

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Variants At 6q21 Associated with Radiation-Induced Second Malignancies After Hodgkin Lymphoma Differentially Regulate PRDM1 Expression Following Radiation Exposure

Blood ◽

10.1182/blood.v118.21.431.431 ◽

2011 ◽

Vol 118 (21) ◽

pp. 431-431

Author(s):

Kenan Onel ◽

Timothy Best ◽

Shan Yu

Keyword(s):

Radiation Exposure ◽

Hodgkin Lymphoma ◽

Genome Wide Association Study ◽

Conflicts Of Interest ◽

Independent Set ◽

Genomic Region ◽

Luciferase Reporter ◽

Malignant Neoplasms ◽

Luciferase Reporter Gene ◽

A Genome

Abstract Abstract 431 Childhood Hodgkin lymphoma (HL) survivors are particularly susceptible to second malignant neoplasms (SMNs) caused by radiation therapy (RT). To identify genetic risk factors for SMNs, we undertook a genome-wide association study of 96 SMN cases and 86 SMN-free controls. Both cases and control sets consisted of long-term survivors of childhood HL who received antecedent RT. Two variants at chromosome 6q21 intergenic between PRDM1 and ATG5 were associated with SMNs (rs4946728: P = 1.26×10−9, OR = 3.17 [2.16–4.65] and rs1040411: P = 4.68×10−8, OR= 2.41 [1.76–3.32]). These results were replicated in an independent set of 63 SMN cases and 75 SMN free controls (rs4946728: P =. 003, and rs1040411: P =. 04). The risk variants form a haplotype associated with decreased basal expression of PRDM1, as well as impaired induction of the PRDM1 protein by radiation exposure. An analysis of the genomic region spanned by the SMN-associated haplotype revealed four evolutionarily conserved putative enhancers, characterized by both H3K27 acetylation and DNase I hypersensitivity. To determine whether these putative enhancers modulate expression following radiation exposure, we cloned each into the pGL3 vector downstream of a luciferase reporter gene. We then transiently transfected each construct into lymphoblastoid cell lines. Data will be presented showing how carriage of either the risk or protective haplotype alters the activity of these enhancers in response to radiation. Furthermore, ChIP-Seq data indicate that NFkB variably binds each putative enhancer. As NFkB has previously been shown to induce PRDM1 expression and is itself activated by radiation, we treated LCLs with an NFkB inhibitor to determine whether NFkB is involved in induction of PRDM1 following radiation exposure. These data demonstrate that incorporating etiologic exposures, such as radiation, can enhance the discovery disease-associated genetic variation and guide functional follow-up experiments. Disclosures: No relevant conflicts of interest to declare.

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Overexpression Of Mir-125a In Bone Marrow CD34+ cells Of Patients With Myelodysplastic Syndrome Is Correlated To a Poor Prognosis and May Contribute To The Pathogenesis Of The Disease Through The Modulation Of NF-Kb Activation and Enhancement Of Differentiation Arrest

Blood ◽

10.1182/blood.v122.21.5206.5206 ◽

2013 ◽

Vol 122 (21) ◽

pp. 5206-5206

Author(s):

Irene Ganan-Gomez ◽

Yue Wei ◽

Hui Yang ◽

Maria Carmen Boyano-Adánez ◽

Guillermo Garcia-Manero

Keyword(s):

Bone Marrow ◽

Overall Survival ◽

Conflicts Of Interest ◽

Fine Tuning ◽

Luciferase Reporter ◽

Mirna Cluster ◽

Luciferase Reporter Gene ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Gene Assay

Abstract Myelodysplastic syndromes (MDS) are a group of clonal malignancies characterized by impaired proliferation and differentiation of hematopoietic stem cells and precursors. The involvement of toll-like receptor (TLR)-mediated signalling in the modulation of myeloid differentiation and its participation in the pathogenesis of MDS are well documented (Wei et al 2013). Increased signaling through this pathway leads to the constitutive activation of NF-kB, which regulates the production of cytokines and mediates cell proliferation and apoptosis (Starczynowski 2010). In addition to the expression of proteins involved in inflammation, the TRL pathway also induces the expression of microRNAs (miRNAs) which participate in the fine-tuning of the inflammatory response (Kawai and Akira 2010). miR-125a and miR-125b are known modulators of hematopoiesis (Gerrits et al. 2012) and have been reported to be involved in several lymphoid and myeloid diseases. Little is known about their role in the pathogenesis of MDS. Interestingly, NF-kB-activating ability has been described for both miR-125a/b (Kim et al. 2012), and miR-125b appears to be upregulated by NF-kB within a positive feedback loop (Zhou et al. 2009; Tan et al. 2012). The aim of this work was to analyze the expression of miR-125a/b in MDS CD34+ cells and to study their relationship with the TLR pathway and differentiation. For this purpose, we analyzed the expression of miR-125a/b by qPCR in bone marrow CD34+ cells of 48 MDS patients, compared it with expression in healthy donors and studied the correlation with overall survival. In our study, we included miR-99b, which is clustered with miR-125a in the genome. Levels of TLR pathway components were detected by qPCR and correlated to those of the miRNAs. Activation of NF-kB was determined in Meg-01 and KG1 cells by the luciferase reporter gene assay, using a vector containing NF-kB responsive elements. Differentiation was studied in K562 and MDS-L cells through colony formation assays combined with analyses of the expression of specific markers by qPCR. For these experiments we used miRNA analogs and a miR-125a anti-sense oligonucleotide. Our results showed that miR-125a, but not miR-125b, is strongly overexpressed in MDS patients (∼15-fold of controls; P<0.01) and that miR-125a levels are significantly and negatively correlated to overall survival of MDS patients (P<0.05). Moreover, expression of miR-99b is also directly connected to the progression of the disease (P<0.05). Both miR-125a and miR-99b cooperate in vitro in the activation of NF-kB (P<0.001); however, we observed a negative correlation between miR-99b/miR-125a expression and levels of TLR2, TLR7 and their downstream proteins MyD88 and JMJD3 (P<0.05), suggesting that NF-kB activation by the miRNA cluster occurs in the absence of TLR signaling. Furthermore, we observed a ∼4-fold increase in NF-kB activity after miR-125a inhibition in the presence of a TLR2 agonist (P<0.001), indicating that miR-125a acts as an NF-kB inhibitor upon TLR stimulation. These results show that miR-125a is involved in the fine-tuning of NF-kB activity and that its effects may depend on the status of the TLR pathway. We then investigated the role of miR-125a in hematopoiesis and found that this miRNA contributes to the blockade of differentiation in the cell lines studied. Therefore, miR-125a could be involved in the pathogenesis or progression of MDS through the modulation of NF-kB activity and differentiation arrest. Thus, this miRNA could be a good prognostic marker and is a potential therapeutic target in MDS. Disclosures: No relevant conflicts of interest to declare.

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Lymphocyte signaling and activation by the CARMA1-BCL10-MALT1 signalosome

Biological Chemistry ◽

10.1515/hsz-2016-0216 ◽

2016 ◽

Vol 397 (12) ◽

pp. 1315-1333 ◽

Cited By ~ 28

Author(s):

Isabel Meininger ◽

Daniel Krappmann

Keyword(s):

Current Knowledge ◽

Inflammatory Diseases ◽

Critical Role ◽

Antigen Receptor ◽

Lymphocyte Activation ◽

Downstream Signaling ◽

Antigen Receptor Signaling ◽

Autoimmune And Inflammatory Diseases ◽

And Function

Abstract The CARMA1-BCL10-MALT1 (CBM) signalosome triggers canonical NF-κB signaling and lymphocyte activation upon antigen-receptor stimulation. Genetic studies in mice and the analysis of human immune pathologies unveiled a critical role of the CBM complex in adaptive immune responses. Great progress has been made in elucidating the fundamental mechanisms that dictate CBM assembly and disassembly. By bridging proximal antigen-receptor signaling to downstream signaling pathways, the CBM complex exerts a crucial scaffolding function. Moreover, the MALT1 subunit confers a unique proteolytic activity that is key for lymphocyte activation. Deregulated ‘chronic’ CBM signaling drives constitutive NF-κB signaling and MALT1 activation, which contribute to the development of autoimmune and inflammatory diseases as well as lymphomagenesis. Thus, the processes that govern CBM activation and function are promising targets for the treatment of immune disorders. Here, we summarize the current knowledge on the functions and mechanisms of CBM signaling in lymphocytes and how CBM deregulations contribute to aberrant signaling in malignant lymphomas.

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Nuclear signalling by Rac GTPase: essential role of phospholipase A2

Biochemical Journal ◽

10.1042/bj3260333 ◽

1997 ◽

Vol 326 (2) ◽

pp. 333-337 ◽

Cited By ~ 22

Author(s):

Byung-Chul KIM ◽

Jae-Hong KIM

Keyword(s):

Phospholipase A2 ◽

Critical Role ◽

Specific Inhibitor ◽

Major Product ◽

Luciferase Reporter ◽

Dependent Manner ◽

Luciferase Reporter Gene ◽

Inhibitory Protein ◽

Rac Gtpase

Rac, one member of Rho family GTPases, stimulates c-fos serum response element (SRE)–luciferase reporter gene in Rat-2 fibroblast cells. By transient transfection analysis, we demonstrated that the activation of phospholipase A2 (PLA2) and the subsequent production of arachidonic acid (AA) are essential for Rac-induced c-fos SRE activation, implying a critical role for PLA2 in the Rac-signalling pathway to the nucleus. Either pretreatment with mepacrine, a specific inhibitor of PLA2, or co-transfection with the expression plasmid of lipocortin-1, a proposed inhibitory protein of PLA2, selectively abolished RacV12-induced SRE activation. Further, we demonstrated that subsequent metabolism of AA, a major product of Rac-activated PLA2, by lipoxygenase (LO) is essential for Rac-induced c-fos SRE activation. In agreement with the role of the PLA2–AA–LO cascade as a potential mediator of Rac signalling to the nucleus, the addition of exogenous AA stimulated c-fos SRE-luciferase activity in an LO-dependent manner. Together, our results demonstrate that ‘Rac-activated PLA2 and subsequent AA metabolism by LO’ constitute a novel and specific pathway in Rac GTPase-induced c-fos SRE activation.

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Mirs-138 and -424 Control Palmitoylation-Dependent CD95-Mediated Cell Death By Targeting Acyl Protein Thioesterases 1 and 2 in Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood.v124.21.1953.1953 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1953-1953

Author(s):

Valeska Berg ◽

Marion Rusch ◽

Nachiket Vartak ◽

Christian Juengst ◽

Astrid Schauss ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Protein Transport ◽

Target Genes ◽

Conflicts Of Interest ◽

Mrna Level ◽

Resonance Energy ◽

Lymphocytic Leukemia ◽

Luciferase Reporter ◽

Fluorescence Lifetime Imaging ◽

Post Translational Modification

Abstract Introduction: Resistance towards CD95-mediated apoptosis is a hallmark of many different malignancies, like it is known from primary chronic lymphocytic leukemia (CLL) cells. Moreover, apoptosis mediated through CD95 is an essential mechanism to eliminate e.g. auto-reactive or virally infected cells. However, its mode of action is still not fully understood. Recently, it could be shown that palmitoylation of CD95 can influence its signaling properties. Nevertheless, the role and regulation of palmitoylated CD95 still needs to be determined. Methods and results: Previously, we could show that miR-138 and -424 are down-regulated in CLL cells. By applying luciferase reporter assays, mutations of the binding sites qRT-PCR and immunoblots after transfection of both miRs, we identified two new target genes, namely acyl protein thioesterase (APT) 1 and 2, which are under control of both miRs and thereby are significantly over-expressed in CLL cells. Interestingly, our data reveal that expression of APTs is already controlled by miRs on mRNA level. This way APT1 is regulated by miR-138 and expression of APT2 is controlled by miR-424. So far, APTs are the only enzymes known to promote de-palmitoylation. Indeed, membrane proteins are significantly less palmitoylated in CLL cells compared to normal B cells as we determined by click-chemistry, which is a non-radioactive method to determine palmitoylated proteins. Importantly, via acyl-biotin exchange assays with subsequent immunoprecipitation of CD95 and fluorescence lifetime imaging microscopy (FLIM) to Foerster resonance energy transfer (FRET) in living cells we identified APTs to directly interact with CD95 to promote de-palmitoylation, thus impairing apoptosis mediated through CD95. As proof of concept APTs were inhibited specifically by siRNAs, miRs-138/-424 or our pharmacological inhibitor Palmostatin B. Thereby we could restore CD95-mediated apoptosis in CLL cells and other cancers, pointing to a central regulatory role of APTs in CD95 apoptosis. Conclusion: The identification of the de-palmitoylation reaction of CD95 by APTs as a miRNA target provides a novel molecular mechanism how malignant cells escape from CD95-mediated apoptosis. Here, we introduce palmitoylation as a novel post-translational modification in CLL. In light of global palmitoylome studies, which show that potentially palmitoylated proteins are involved in all central cellular processes, such as protein transport, survival, migration, apoptosis and B-cell receptor signaling, this emphasizes the importance of palmitoylation and might put it on par with modifications like phosphorylation. Disclosures No relevant conflicts of interest to declare.

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AML1-ETO Represses Multiple Transcription Factor Induced CTSG Activation by Reducing H3 Acetylation

Blood ◽

10.1182/blood.v122.21.3750.3750 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3750-3750

Author(s):

Yun Tan ◽

Wen Jin ◽

Kang Wu ◽

Kankan Wang

Keyword(s):

Transcription Factor ◽

Transcription Factors ◽

Target Genes ◽

Conflicts Of Interest ◽

Critical Role ◽

Negative Regulator ◽

Cathepsin G ◽

Luciferase Reporter ◽

Aberrant Expression ◽

H3 Acetylation

Abstract Acute myeloid leukemia (AML) is often accompanied with the aberrant expression of transcription factors. In t(8;21) AML, the AML1-ETO fusion protein executes its critical role in leukemogenesis through the interference with hematopoietic transcription factors (TFs) including AML1, C/EBPα, PU.1 and c-Myb. These transcription factors cooperate to modulate hematopoiesis by regulating their differentiation-related target genes. In our previous work, we have identified that AML1-ETO suppresses the AML1-dependent transactivation of the gene encoding the neutrophil granule protease, cathepsin G (CTSG). However, the detailed mechanisms of AML1-ETO mediated transrepression, especially coordinated regulation of hematopoietic transcription factors, have not been characterized yet. To investigate the regulatory pattern of CTSG by hematopoietic specific transcription factors, we constructed a luciferase reporter containing the CTSG promoter and co-transfect it with AML1, c-Myb, C/EBPα or PU.1 to 293T cells. The results of luciferase assays showed that these TFs individually activated the CTSG promoter, and synergistic transactivation occurred between AML1 and c-Myb, C/EBPα and PU.1, and PU.1 and c-Myb on the CTSG promoter. Furthermore, AML1/ETO effectively suppressed the transcription factor-dependent transactivation and synergistic transactivation of the CTSG promoter. Chromatin immunoprecipitation assays further demonstrated that AML1-ETO coexisted with these TFs on the CTSG promoter in AML1/ETO-positive Kasumi-1 cell line, indicating AML1-ETO was tethered to the chromatin bound by these TFs. The data suggested that AML1-ETO might act as a negative regulator by interfering the normal function of hematopoietic TFs instead of competing for their binding. In addition, to reveal the underlying mechanism of AML1/ETO-mediated transcription repression at the epigenetic level, we examined the epigenetic status of the CTSG promoter in AML1-ETO negative and positive cells, and found the level of histone H3 Lys9 acetylation on the CTSG promoter was obviously lower in AML1-ETO positive cells than that in AML1-ETO negative cells. The data suggested that AML1-ETO might repress the gene transcription by changing the H3 acetylation status of its target gene. Collectively, our findings demonstrate that AML1-ETO represses the transactivation of the CTSG promoter mediated by multiple hematopoietic transcription factors through a decrease of H3 acetylation. Disclosures: No relevant conflicts of interest to declare.

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GDF-5 promotes epidermal stem cells proliferation via Foxg1-cyclin D1 signaling

Stem Cell Research & Therapy ◽

10.1186/s13287-020-02106-7 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Xiaohong Zhao ◽

Ruyu Bian ◽

Fan Wang ◽

Ying Wang ◽

Xue Li ◽

...

Keyword(s):

Stem Cells ◽

Wound Healing ◽

Cyclin D1 ◽

Western Blotting ◽

Target Genes ◽

Critical Role ◽

Luciferase Reporter ◽

Epidermal Stem Cells ◽

Luciferase Reporter Gene ◽

Partial Thickness Burn

Abstract Objective Epidermal stem cells (EpSCs) can self-renew, which are responsible for the long-term maintenance of the skin, and it also plays a critical role in wound re-epithelization, but the mechanism underlying EpSCs proliferation is unclear. GDF-5, also known as BMP-14, is a member of the BMP family and can be used as a self-renewal supporter. Here, we studied the effects of GDF-5 on mouse EpSCs proliferation mechanism in wound healing. Methods Firstly, the effects of GDF-5 on EpSCs proliferation was tested by using CCK8 reagent and PCNA expression was analyzed by Western blotting. Secondly, we screened genes that promote EpSCs proliferation in the FOX and cyclin family by qPCR, and then the protein expression level of the selected genes was further analyzed by Western blotting. Thirdly, siRNA plasmids and pAdEasy adenovirus were transfected or infected, respectively, into mouse EpSCs to detect the effect of target genes on GDF-5-induced cell proliferation. Furthermore, we injected GDF-5 to a deep partial thickness burn mouse model for finding out whether EpSCs proliferation can be detected by immunohistochemical. Finally, the relevant target genes were analyzed by qPCR, immunoblotting, and dual-luciferase reporter gene detection. Results We discovered that 100 ng/ml recombinant mouse GDF-5 was the optimal concentration for promoting mouse EpSCs proliferation. Through preliminary screened by qPCR, we found that Foxg1 and cyclin D1 could be the downstream molecules of GDF-5, and the results were confirmed by Western blotting. And the effect of GDF-5 on mouse EpSCs proliferation was adjusted by Foxg1/cyclin D1 in vitro and in vivo. Besides, GDF-5-induced transcription of cyclin D1 was regulated by Foxg1-mediated cyclin D1 promoter activity. Conclusion This paper showed that GDF-5 promotes mouse EpSCs proliferation via Foxg1-cyclin D1 signal pathway. It is suggested that GDF-5 may be a new approach to make EpSCs proliferation which can be used in wound healing.

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H3K36 Trimethylation Mediated By SETD2 Regulates Cell Proliferation and Cell Cycle By Modulating BCMA-JNK and c-Myc Pathways in Multiple Myeloma

Blood ◽

10.1182/blood-2021-145120 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2200-2200

Author(s):

Pan Zhou ◽

Mengyu Xiao ◽

Yanliang Bai ◽

Huixia Cao ◽

Weiping Yuan ◽

...

Keyword(s):

Cell Cycle ◽

Multiple Myeloma ◽

Cell Proliferation ◽

Conflicts Of Interest ◽

Critical Role ◽

Mapk Signaling ◽

Gene Set Enrichment Analysis ◽

Luciferase Reporter ◽

Brdu Incorporation ◽

Down Regulation

Abstract In addition to genetic aberrations, accumulating evidence indicates that deregulation of histone methyltransferases, such as MMSET, EZH2 and KDM6A, plays crucial roles in the oncogenic transformation and development of multiple myeloma (MM). For example, overexpression of MMSET leading to a global increase in H3K36me2, is believed to be the driving force in the pathogenesis of t (4;14) MM. However, as the histone methyltransferase is responsible for H3K36me3, the role of SETD2 is not been known in myeloma. To explore the possible clinical value of SETD2 in MM, we first examined the gene expression profile from GEO database, which indicated that the SETD2 expression was significantly decreased in MM patients when compared with monoclonal gammopathy of undetermined significance and smoldering multiple myeloma patients (GSE6477). Moreover, the expression of SETD2 decreased with the advanced international staging system stage of MM patients (GSE19784). The Kaplan-Meier analysis showed that low expression of SETD2 was significantly associated with a poor overall survival in MM patients (GSE2658, P<0.05; GSE9782, P<0.001). Thus, our analysis suggests that SETD2 might participate in cancer progression and could become a biomarker for the prognosis of patients with MM. We then investigated the biological role and the underlying mechanism of SETD2 in MM. Firstly, we used lentiviral-mediated RNA interference to knockdown SETD2 (SETD2 KD) in MM cell lines (RPMI8226 and MM.1S). CCK8 and colony-forming assays showed that reduced expression of SETD2 significantly promoted MM cell proliferation and colony growth. BrdU incorporation assay revealed increased DNA synthesis in SETD2 KD MM cells. Then, treatment with JIB-04, a small molecule inhibitor targeting H3K36me3 loss in SETD2 KD MM cells showed that H3K36me3 recovery was capable of reversing the tumor-promoting effect due to SETD2 down-regulation of MM cells in vitro. Moreover, the xenograft growth assay revealed that SETD2 down-regulation facilitated tumor growth and JIB-04 treatment exhibited anti-myeloma activity in vivo. Therefore, we conclude that SETD2 plays an important role in MM maintenance, and inhibition of H3K36me3 shows therapeutic efficacy for MM. To further explore the underlying mechanisms, we performed the RNA-Seq analysis and discovered that low H3K36me3 level was associated with reduced expression of CDKN1A and increased expression of TNFRSF17 (BCMA) and c-Myc in MM cells. The Gene Set Enrichment Analysis (GSEA) revealed that MAPK signaling pathway was enriched in SETD2 knockdown and JIB-04 treated MM cells. Subsequent Western blotting analysis further confirmed that SETD2 KD cells had increased JNK activation, while JIB-04 treated group showed decreased level of p-JNK. To investigate whether BCMA was the directly transcriptional target for H3K36me3, we carried out a dual-luciferase reporter assay in both SETD2 KD and JIB-04 treated MM cells, and confirmed that H3K36me3 inhibited the expression of BCMA through physically interacting with motifs in its promoter. Furthermore, down-regulation of BCMA in SETD2 KD MM cells could lead to a reduction of p-JNK and an up-regulation of CDKN1A, and resulting inhibited cell proliferation and cell cycle progression. Furthermore, blockage of the JNK pathway by its inhibitor SP600125 resulted in significant inhibition of MM cell proliferation induced by SETD2 knockdown. Additionally, suppression of c-Myc using 10058-F4 inhibited proliferation, and induced cell cycle arrest as well as CDKN1A expression in SETD2 KD MM cells. These results indicate that BCMA/JNK and c-Myc pathways were involved in STED2 and H3K36me3 mediated cell proliferation in MM. Together, our data delineate that SETD2-dependent H3K36me3 modification plays a critical role in regulation cell proliferation and cell cycle by BCMA-JNK and c-Myc pathways in MM cells. Targeting the SETD2-H3K36me3 pathway represents a promising therapy for MM. Disclosures No relevant conflicts of interest to declare.

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