Dysregulated Apurinic/Apyrimidinic Endonucleases (Ape1 and Ape2) Lead to Genetic Instability in Multiple Myeloma.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1418-1418
Author(s):  
Masood A. Shammas ◽  
Hemant Koley ◽  
Sima Shah ◽  
Ramesh B. Batchu ◽  
Pierfrancesco Tassone ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Homologous Recombination ◽  
Genomic Instability ◽  
Cell Lines ◽  
Plasma Cells ◽  
Dna Recombination ◽  
Endonuclease Activity ◽  
Single Nucleotide ◽  
Genetic Changes

Abstract Multiple myeloma (MM) is associated with significant genomic instability. Homologous recombination (HR), which is elevated in MM, is considered to be responsible for this instability. As endonucleases play an important role in mediating HR, here we have evaluated the role of endonuclease in biology and progression of MM. Gene expression profile using Affymetrix U133 array showed > 2 fold elevation of Ape1 or Ape2 or both in 5 of 6 MM cell lines and 12 of 15 patient samples. Immunocytochemistry confirmed upregulation of Ape1 protein in MM cell lines. A Plasmid degradation assay confirmed significantly elevated endonuclease activity in MM cells compared to normal plasma cells. To identify the pre-dominating endonuclease activity, the degradation assay was carried out in the presence of specific endonuclease inhibitors. Harmane and methoxyamine (MA), specific inhibitors of apurinic/apyrimidinic endonucleases effectively inhibited significant endonuclease activity, while other endonuclease inhibitors ACPD and FK506 had minimal effects, confirming predominant role of apurinic/apyrimidinic endonucleases (APE) in mediating increased endonuclease activity in MM. We investigated the role of elevated APE endonuclease activity on DNA recombination and subsequent genomic re-arrangements. Using a plasmid-based assay we have previously demonstrated significantly elevated homologous recombination (HR) in MM. Inhibition of endonuclease by methoxyamine suppressed HR activity by 85 ± 2% in MM cells. Next, we evaluated whether inhibition of HR by methoxyamine can affect the frequency of acquisition of new genetic changes in MM cells using single nucleotide polymorphism (SNP) arrays (Affymetrix) as indicator of genomic instability. In three independent experiments, methoxyamine reduced the acquisition of new loss of heterozygocity (LOH) loci by an average of 71%. These data suggest that the dysregulated APE endonucleases contribute significantly to the genomic instability, acquisition of new mutations and progression of MM and provides the rationale for targeting endonuclease activity to prevent disease progression including development of drug resistance.

Molecular Basis of Genomic Instability and Progression in Multiple Myeloma: Potential Role of Apurinic (Apyrimidinic) Endonuclease.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1561-1561
Author(s):  
Masood A. Shammas ◽  
Hemanta Koley ◽  
Paola Neri ◽  
Pierfrancesco Tassone ◽  
Ramesh B. Batchu ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Genomic Instability ◽  
Cell Lines ◽  
Molecular Basis ◽  
Endonuclease Activity ◽  
Ap Endonuclease ◽  
Myeloma Cells ◽  
Genome Wide ◽  
New Mutations

Abstract Genetic instability is a prominent feature of most cancers including multiple myeloma (MM) and is responsible for ongoing accrual of mutational changes which may lead to development of drug resistance and metastasis. The molecular basis for the generation of genetic diversity in MM is therefore extremely important to understand carcinogenesis and to identify novel targets for treatment. As genomic rearrangements require excision of DNA, we hypothesized that an elevated endonuclease activity may induce recombination and subsequent genomic instability in cancer cells. We developed a plasmid degradation assay that confirmed significantly elevated endonuclease activity in MM cells compared to normal plasma cells. To identify the pre-dominating endonuclease the degradation assay was carried out in the presence of specific endonuclease inhibitors, which identified apurinic/apyrimidinic endonuclease (Ape1 and Ape2) as the predominant endonucleases in mediating increased endonuclease activity in MM. Gene expression analysis confirmed > 2 fold elevation of Ape1 or Ape2 or both in 5 of 6 MM cell lines and 12 of 15 patient samples. Both immunocytochemistry and western blot analyses confirmed upregulation of Ape1 protein in all MM cell lines and patient samples. Next, we investigated the role of elevated APE endonuclease activity in DNA recombination and subsequent genomic re-arrangements. Using a plasmid-based assay we have previously demonstrated significantly elevated homologous recombination (HR) in MM. To investigate the role of elevated AP endonuclease activity in MM, we cultured myeloma cells in the presence of methoxyamine (MX), which specifically inhibits AP endonuclease activity, and evaluated its effect on HR activity and genome-wide appearance of new mutations. Exposure of intact myeloma cells to MX resulted in > 90% inhibition of HR activity and a significant (71±10.9%; p<0.05) reduction in the appearance of new mutations compared to untreated cells, as assessed by genome-wide loss of heterozygosity (LOH) assay (Affymetrix). We also evaluated the effects of overexpression of Ape1 & 2 in normal fibroblasts which have low endonuclease activity. The transgenic upregulation of AP endonucleases (Ape1 and Ape2) in normal cells led to a significant increase in the lecombination activity, leading to a marked mutational instability as indicated by the appearance of over 20,063 and 20,143 new LOH loci per 100,000 polymorphic regions examined throughout the genome, at population doublings 25 and 50 respectively. Mutational instability was also associated with chromosomal instability confirmed by spectral karyotyping of these cells showing significant numerical and structural chromosomal abnormalities. These changes were associated with indefinite growth of cells and formation of tumors when injected in SCID mice. These data suggest that elevated AP endonuclease may be responsible for mutational and chromosomal instabilities, leading to progression of myeloma.

Microrna Expression Profile Identifies Distinct Clinically Relevant Sub-Groups in Multiple Myeloma: Novel Prognostic Markers and Potential Targets for Therapy

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 96-96 ◽  
Author(s):  
Sophia Adamia ◽  
Herve AvetLoiseau ◽  
Samirkumar B Amin ◽  
Yu-Tzu Tai ◽  
Steven P. Treon ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Differentiation ◽  
Clinical Outcome ◽  
Signaling Pathways ◽  
Cell Lines ◽  
Plasma Cells ◽  
Normal Plasma ◽  
Group A ◽  
Group B

Abstract MicroRNA, an abundant class of small endogenous RNAs, regulate target genes through inducing translational inhibition and cleavage of targeted transcripts. To date, microRNAs have been implicated in normal biological processes, including development, cell differentiation, apoptosis and proliferation as well as in malignant transformation. However, their role in multiple myeloma (MM) remains unknown. Here we investigated role of microRNAs in myelomagenesis, and their influence on prognosis and clinical outcome. We evaluated profiles of 384 microRNAs in bone marrow derived CD138+ plasma cells (PC) from 79 uniformly treated MM patients, 11 MM cell lines and 9 healthy donors using qRT-PCR based microRNA array. The relative expression was calculated using comparative Ct method, and data was normalized using endogenous controls and analyzed using SDS, RQ manager, R and dChip softwares. MicroRNA expression profiles detected in MM patients were correlated with clinical outcome measures. We observed significant modulate expression of 61 microRNAs in myeloma cells compared to normal plasma cells. When more stringent criteria were used, we identified 24 differentially expressed microRNAs in patient samples. Further, unsupervised hierarchical clustering of filtered microRNAs, based on their DCt values, identified two major groups within the MM population (groups A and group B). Samples of Group A clusters with MM cell lines, indicating more proliferative nature of MM patient cells. Within B group, a second degree node group B2, clusters with normal plasma cells indicating more indolent course, while patients in an additional node B1 represented an assorted pattern. The unsupervised clustering of all MM samples showed consistent changes in miR-30b, -30c, -30d, -142-5p, -24, -191, -181d, -374, -146b, -140, -145, -125a, -151, -223, -155, let7b, indicative of a role of these microRNA in myelomagenesis; while supervised analysis of samples within groups A and B identified modulated expression of different sets of miRNAs. In group A miR-585 and let-7f were upregulated 8–12 fold, while miRs -125a, -126, -155, -223, -146a, -374 -19a, -20a, -26a, -30a -5p, -30b, and -30d were significantly downregulated; in group B, all differentially expressed microRNAs were downregulated (p<0.001) compared to normal plasma cells. These modulated miRNAs target critical signaling pathways including apoptosis, hematopoietic cell differentiation and proliferation, survival and angiogenesis by upregulating function of HOX9, c-myc, VCAM-1, Bcl-2, E2F1, SHP1, SHP2, VEGF, and DUSp6 molecules. We further analyzed the effect of microRNA on clinical outcome. We have observed significantly superior event free and overall survival of patients in group B2 compared to patients in group A (2 yr estimated EFS 79% versus 54% respectively; p=0.05; and 2 yr estimated OS 94% versus 70% respectively; p =0.017). Taken together this data identifies critical microRNAs as modulators of gene expression and signaling pathways and provides potential novel microRNA and gene targets in MM to both understand biological behavior and for therapeutic application.

scholarly journals DNA Methyltransferase Inhibitors Increase Expression of CD38 and Enhance Daratumumab Efficacy in Multiple Myeloma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5618-5618 ◽  
Author(s):  
Priya Choudhry ◽  
Margarette C. Mariano ◽  
Arun P Wiita
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Cell Lines ◽  
Research Funding ◽  
Plasma Cells ◽  
Cpg Island ◽  
Ex Vivo ◽  
Single Agent ◽  
Cd38 Expression

Abstract Introduction: The anti-CD38 monoclonal antibody Daratumumab is highly effective against multiple myeloma, is well tolerated, and has high single agent activity as well as combination effects with lenalidomide-dexamethasone as well as bortezomib-dexamethasone. Patient response to daratumumab monotherapy is highly correlated with pretreatment levels of CD38 expression on MM plasma cells (Nijhof et al, Leukemia (2015) 29:2039) and CD38 loss is correlated with daratumumab resistance (Nijhof et al, Blood (2016) 128:959). As a result, there is significant interest in elucidating the regulation and role of CD38 in MM. Recently, All Trans Retinoic Acid (ATRA), a known small molecule inducer of CD38 in myeloid cells, as well as the FDA-approved histone deacetylase inhibitor panobinostat, were both demonstrated to induce CD38 in MM plasma cells leading to increased lysis by daratumumab. Examining ENCODE data, we found the presence of a CpG island at the first exon of CD38. We hypothesized that removing methylation sites from this CpG island may de-repress CD38 transcription and lead to increased CD38 protein at the cell surface in MM plasma cells. Therefore, here we studied the role of DNA methyl-transferase inhibitors (DNMTis), currently FDA-approved for treatment of myelodysplastic syndrome, as agents to potentiate daratumumab therapy. Methods: We treated MM cell lines (RPMI-8226, MM.1S, XG-1, KMS12-PE) with two different DNMTis, 5-Azacytidine and decitabine, and assessed CD38 cell surface expression by flow cytometry. Similarly, we treated MM patient bone marrow aspirates ex vivo and assessed induction of CD38 expression in the CD138 positive population by flow cytometry. We analyzed CD38 mRNA levels and total CD38 protein levels by qRT-PCR and western blotting respectively. ATRA was used as a positive control in all experiments. We further tested the functional effect of DNMTi treatment on MM cell lines using an Antibody Dependent Cell Cytotoxicity (ADCC) assay. Briefly, live treated cells were incubated overnight with daratumumab and NK92-CD16 transgenic cells at and E:T ratio of 20:1, and lysis was measured using CytoTox-Glo (Promega). Results: Flow analysis revealed that DNMTi treatment induces a 1.2-2 fold increase in CD38 surface protein expression in a dose-dependent manner across MM cell lines. DNMTi treatment consistently yielded similar or higher increases in CD38 expression than that seen in ATRA- or panobinostat-treated cells. Despite significantly lower single-agent cytotoxicity, we found that decitabine led to similar surface CD38 induction as 5-Azacytidine. By RT-qPCR, 5-Azacytidine increased CD38 mRNA expression ~3 fold versus DMSO control, compared to ~2 fold mRNA increase with ATRA. In functional ADCC assays, DNMTi treatment also led to greater lysis than ATRA. Furthermore, the combination of both DNMTi and ATRA was additive, leading to the greatest lysis by NK cells. In contrast, in ex vivo-treated patient samples, ATRA induced greater CD38 expression than 5-Azacytidine on malignant plasma cells. However, this result is expected since MM plasma cells from patients typically do not proliferate in standard ex vivo culture, and active DNA replication is a requirement for successful DNMT inhibition based on known mechanism of action. In patients, however, we anticipate that continual plasma cell proliferation will lead to effective increases in CD38 after DNMTi treatment, as found in MM cell lines here. Summary and Conclusions: Our results here demonstrate that CD38 expression in MM cells is regulated by DNA methylation and targeting DNMTs with small molecule inhibitors leads to increased vulnerability to Daratumumab treatment. We propose that combination treatment with DNMTi and Daratumumab can lead to higher efficacy of daratumumab in daratumumab-naïve MM, as well as reversal of daratumumab-resistance. These combinations should be tested in clinical trials. Disclosures Wiita: Sutro Biopharma: Research Funding; TeneoBio: Research Funding.

Identification of EpHA3 As a New Potential Molecular Target in Multiple Myeloma.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2926-2926
Author(s):  
Antonella Caivano ◽  
Francesco La Rocca ◽  
Alessandra Favole ◽  
Sonia Carturan ◽  
Enrico Bracco ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Monoclonal Antibody ◽  
Cell Lines ◽  
Plasma Cells ◽  
Molecular Target ◽  
Over Expression ◽  
In Vitro Angiogenesis ◽  
Novel Therapeutic

Abstract Abstract 2926 Introduction Angiogenesis plays a central role in the progression of both solid and hematological tumors. In particular, in multiple myeloma (MM) the critical role of bone marrow (BM) microenvironment and angiogenesis has been well documented. The past decade has witnessed a dramatic improvement in the therapeutic options in MM. However, the disease remains incurable, underscoring the need for continued efforts towards understanding MM biology and exploitation of novel therapeutic approaches. In this setting, monoclonal antibodies against myeloma-specific cell surface antigens represent a promising therapeutic approach, which is however hampered by a lack of appropriate target structures expressed across all pathogenic myeloma cells. The Eph receptors, a large family of receptor tyrosine kinases (RTKs) activated by ephrins binding, have been implicated in many processes involved in malignancy, including alteration of the tumor microenvironment and in angiogenesis, in both of which EpHA3 likely plays an active role. Aberrant expression of EpHA3 is seen in many types of hematolologic malignancies (some leukemic cell lines, T-cell lymphoma, acute lymphoblastic leukemia, myeloproliferative neoplasms) although it is not expressed ubiquitously. Finally, the over-expression of Eph is believed to be sufficient to confer tumorigenic potential although probably further mechanisms can occur to abnormally activate the receptor. Basing on the role of EpHA3 in haematological malignancies, a first-in-class engineered IgG1 antibody targeting the EpHA (KB004) was developed and it is now under phase I clinical trials in USA and Australia for the treatment of EpHA3 overexpressing hematological myeloid malignancies refractory to conventional treatment. We investigated the EpHA3 role and its preferential membrane–bound by GPI linker ligand EFNA5, in MM patients in order to define EpHA3 as new molecular target for a novel therapeutic approach with a specific anti EpHA3 monoclonal antibody. The EpHA3 expression has been studied through a comparative proteomic analysis between BM endothelial cells (ECs) of patients with MM (MMECs) or with monoclonal gammopathy of undetermined significance (MGECs), of control subjects (normal ECs) and in MM cell lines. Methods After written informed consent, BM aspirates have been collected from 20 MM and 4 MGUS patients. Normal ECs were derived from 3 BM aspirates of subjects with anemia due to iron or vitamin B12 deficiency. We analyzed the expression levels of EpHA3 in normal ECs, MGECs and MMECs and MM cell lines evaluating the mRNA and protein levels by RT-qPCR and by WB coupled to ImmunoFluorescence analysis. The biological effects of EpHA3 targeting in MMECs have been studied silencing the EpHA3 mRNA in MMECs and testing them at 72h after silencing in series of functinal assays including viability assay by trypan blue exclusion staining and by in vitro angiogenesis assay followed by measurement of mesh areas and vessel length. Moreover, we studied EFNA5 mRNA expression levels in Normal ECs, MGECs and MMECs and in MM cell lines by PCR. Results Our data showed that EpHA3 mRNA levels are progressively increased from ECs to MGECs reaching the highest values in MMECs. Subsequent analysis by WB and immunofluorescence confirmed EpHA3 protein upregulation among the different EC types. The MMECs in which EpHA3 has been silenced revealed a protein level reduction of approximately 60% when compared to the control. We could not detect major viability defects. Furthermore, in vitro angiogenesis inhibition was marginal when compared to the not silenced counterpart. To know whether EpHA3 may impact not only MM angiogenesis but also plasma cells, three MM cell lines were studied for the EpHA3 expression. We found the plasma cell lines gave constant over expression of EpHA3. Finally, the preliminary data regarding EFNA5 mRNA expression level showed it is expressed in either MMECs and MM plasma cell lines. The evaluation of KB004 effect on MMECs in term of apoptosis induction and in vitro tube formation inhibition, as well as the analysis of EpHA3 levels in primary MM plasma cells are in progress. Conclusions From this study we expect to characterize the role of the EpHA3in MM patients and to provide experimental evidences supporting the possibility of using EpHA3 as a new molecular target for MM by proving the in vitro efficacy of a monoclonal antibody to target the angiogenesis of MM. Disclosures: No relevant conflicts of interest to declare.

Role Of Sirtuin-6 In Maintenance Of Genomic Instability In Multiple Myeloma Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 276-276
Author(s):  
Michele Cea ◽  
Antonia Cagnetta ◽  
Mariateresa Fulciniti ◽  
Yu-Tzu Tai ◽  
Chirag Acharya ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Dna Damage ◽  
Dna Repair ◽  
Genomic Instability ◽  
Cell Lines ◽  
Dsb Repair ◽  
Dna Damaging Agents ◽  
Genotoxic Agents ◽  
Equity Ownership

Abstract Background Deregulation of the DNA damage response (DDR) signaling machinery underlies genomic instability, leading to cancer development and clonal evolution. Multiple Myeloma (MM) remains an incurable disease characterized by a highly unstable genome, with aneuploidy observed in nearly all patients. The mechanism causing this karyotypic instability is largely unknown, but recent observations have correlated these abnormalities with dysfunctional DDR machinery. Mammalian NAD+-dependent deacetylase sirtuin-6 (SIRT6) is emerging as new protein involved in multiple pathways, including maintenance of genome integrity. Methods A panel of 18 MM cell lines, both sensitive and resistant to conventional and novel anti-MM therapies, was used in this study. Blood and BM samples from healthy volunteers and MM patients were obtained after informed consent and mononuclear cells (MNCs) separated by Ficoll-Paque density sedimentation. Patient MM cells were isolated from BM MNCs by CD138-positive selection. Lentiviral delivery was used for expression and knock-down of SIRT6 in MM cell lines. The biologic impact of SIRT6 phenotype was evaluated using cell growth, viability and apoptosis assays. DNA Double-Strand Breaks (DSB) repair occurring via homologous recombination (HR) or non-homologous end-joining (NHEJ) pathways was assessed using a transient direct repeat (DR)-GFP/I-SceI system. Results A comparative gene expression analysis of 414 newly-diagnosed uniformly-treated MM patients showed high levels of SIRT6 mRNA in MM patients versus MGUS or normal donors; moreover, in active MM elevated SIRT6 expression correlated with adverse clinical outcome. Due to its prognostic significance, we further evaluated its role in MM biology. We found higher SIRT6 nuclear expression in MM cell lines and primary cells compared to PBMCs from healthy donors. Targeting SIRT6 by specific shRNA increased MM cell survival by reducing DNA repair efficiency (HR and NHEJ). Whole genome profiling of three different SIRT6 knockout (Sirt6-/-) MM cell lines identified a restricted effect of SIRT6 silencing on transcription of DNA damage genes, which also represented the most down-regulated genes. Consistent with these data, GSEA algorithm revealed that gene set regulating DNA repair were prominently enriched in SIRT6 depleted cells (p<0.0001 and FDR=0.003), confirming the role of SIRT6 in this pathway. We next examined the therapeutic relevance of SIRT6 inhibition in MM by evaluating the effect of SIRT6 depletion on cytotoxicity induced by genotoxic agents. SIRT6 shRNA impaired DNA DSB repair pathways triggered by DNA damaging agents, thereby enhancing overall anti-MM activity of these agents. Finally, in concert with our in vitro data, studies using our human MM xenograft model confirmed that SIRT6 depletion enhanced anti-MM activity of DNA-damaging agents. Conclusion Collectively, our data provide basis for targeting SIRT6 as a novel therapeutic strategy in combination with genotoxic agents to enhance cytotoxicity and improve patient outcome in MM. Disclosures: Tai: Onyx: Consultancy. Hideshima:Acetylon Pharmaceuticals: Consultancy. Chauhan:Vivolux: Consultancy. Anderson:celgene: Consultancy; onyx: Consultancy; gilead: Consultancy; sanofi aventis: Consultancy; oncopep: Equity Ownership; acetylon: Equity Ownership.

scholarly journals Role of N6-Methyladenosine (m6A) RNA Modification in Multiple Myeloma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5601-5601
Author(s):  
Christian Bach ◽  
Magdalena Leffler ◽  
Cindy Flamann ◽  
Jan Kronke ◽  
Dimitrios Mougiakakos ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Messenger Rna ◽  
Plasma Cells ◽  
Conflicts Of Interest ◽  
Stop Codon ◽  
Mrna Levels ◽  
Molecular Abnormalities ◽  
Gene Expression Control

Abstract Multiple myeloma (MM) is considered a chronic and incurable disease due to its highly complex and heterogeneous molecular abnormalities. In recent years, integrating proteasome inhibitors and immunomodulatory drugs into MM frontline therapy has significantly improved treatment efficacy with a median overall survival (OS) being prolonged from 3-4 to 7 years. Despite this progress, patients refractory to the aforementioned agent classes display a median OS of only 9 months. Thus, the clinical necessity for developing novel therapeutic alternative approaches is self-evident. Methylation of N6-adenosine (m6A) is known to be important for diverse biological processes including gene expression control, translation of protein, and messenger RNA (mRNA) splicing. m6A regulatory enzymes consist of "writers" METTL3 and METTL14, "readers" YTHDF1 and YTHDF2, and "erasers" FTO and ALKBH5. However, the functions of m6A mRNA modification and the specific role of these enzymes in MM remain unknown. Here we report that METTL3, a key component of the m6A methyltransferase complex, is highly expressed in MM cell lines and in isolated patient's MM cells. In contrast, we found no significant differences in the expression of the m6A demethylases FTO and ALKBH5. Accordingly, compared to plasma cells from healthy donors, global PolyA+ RNA showed a significant increase in m6A content in patient's MM plasma cells. In MM cell lines, global m6A profiling by methylated RNA-immunoprecipitation sequencing revealed m6A peaks near the stop codon in mRNAs of multiple oncogenes including MAF and CCND1. Cross-linking immunoprecipitation showed that METTL3 bound to the m6A peak within MAF and CCND1 mRNA. Depletion of METTL3 by shRNA had little effect on global mRNA levels, but specifically reduced protein levels of c-Maf and Cyclin D1. Moreover, downregulation of METTL3 in several MM cell lines results in cell cycle arrest and apoptosis. Together, these results describe a role for METTL3 in promoting translation of a subset of oncogenes in MM and identify this enzyme as a potential therapeutic target for multiple myeloma. Disclosures No relevant conflicts of interest to declare.

scholarly journals The Role of PHF19 As a Promoter of Tumorigenicity and Therapeutic Target in Multiple Myeloma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 508-508
Author(s):  
Carolina D. Schinke ◽  
Pingping Qu ◽  
Shmuel Yaccoby ◽  
Valeriy V Lyzogubov ◽  
Veronica MacLeod ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Candidate Gene ◽  
Cell Lines ◽  
Crucial Role ◽  
Plasma Cells ◽  
Total Therapy

Introduction - Multiple Myeloma (MM) is a hematologic malignancy characterized by clonal growth of differentiated plasma cells (PCs). Despite improvement in MM therapy, the disease remains mostly incurable and is characterized by recurrent relapses with development of resistant clones that eventually lead to patient death. The pathways that lead to resistant and aggressive MM are not fully understood highlighting the need to improve our understanding of MM biology to identify potential new pathways and therapeutical targets. PHD Finger Protein 19 (PHF19) is a regulator of Polycomb Repressive Complex 2 (PRC2), the sole methyltransferase complex capable of catalyzing H3K27me3 to induce and enforce gene repression. PRC2 employs enhancer of zeste homolog 1 and 2 (EZH1/EZH2) as enzymatic subunits to hypermethylate H3K27. While overexpression and gain of function mutations of EZH1/2 have been observed in many cancers the role of this particular pathway in MM remains poorly understood. In the present study, we report on PHF19 as a new candidate gene to play a potential crucial role in MM oncogenesis. Methods- Gene expression profiling (GEP; Affymetrix U133 Plus 2.0) was performed on 739 MM patients (from total therapy trials [TT] 3-5; low risk MM n=636, high risk MM n=103), 42 patients with monoclonal gammopathy of undetermined significance (MGUS), 73 smoldering MM patients, 42 patients with primary plasma cell leukemia and 34 healthy donors. Myeloma risk was determined by the GEP 70 signature as previously defined. To test the implications of functional PHF19 knock down (KD) we used TRIPZ inducible PHF19 shRNA vs. scrambled control (Dharmacon) in two MM cell lines (JJN3 and ARP1). Real time PCR as well as western blotting was used to confirm PHF19 KD as well as to elucidate the effect on H3K27me3 (Cell Signaling). Functional in vitro studies included proliferation (Promega), clonogenic assays (StemCell), cell cycle and apoptosis assays (both Invitrogen). In vivo studies were performed using SCID mice that were subjected to tail vain injection with PHF19 KD JJN3 cells (n=10) or scrambled shRNA control (n=10). Weekly ELISA (Bethyl) and in vivo imaging (Xenogen) were performed and survival was recorded. Results- GEP of the previously mentioned patient populations and healthy controls identified PHF19 (chr9q33.2) as a candidate gene that was consistently dysregulated in MM patients. Mean expression levels at different MM stages correlated with disease aggressiveness (ANOVA, p&lt;0.0001), Figure 1. High expression of PHF19 (log2&gt;10.46) at diagnosis correlated significantly with adverse clinical parameters, including ISS III, anemia and elevated LDH, as well as worse overall survival (5 yr OS = 29% for patients with high PHF19 expression vs 77% for patients with low PHF19 expression [log2&lt;10.46], p&lt; 0.0001). These results led us to test the implications of functional PHF19 KD using TRIPZ inducible PHF19 shRNA vs. scrambled control in the JJN3 and ARP1 MM cell lines. PHF19 KD led to a drastic reduction of H3K27me3 thereby resulting in significantly reduced proliferation via cell cycle arrest, while apoptosis was not substantially altered. Clonogenic assays showed a significant reduction in colony numbers and size of MM cells with PHF19 KD compared to the control (&gt;75% reduction in both cell lines, p&lt;0.05). Xenograft studies showed consistently less tumor burden in the mice injected with PHF19 KD cells compared to scrambled control, evident through ELISA testing for IgG Kappa (Median =180 mg/ml for scrambled control vs 80 mg/ml for PHF19 KD at week 8, p=0.07) and bioimaging (Median bioilumisence 2.1x108 p/s for scrambled control vs. 0.8x108 p/s for PHF19 KD at week 8, non-significant). Median OS in mice injected with PHF19 KD cell was substantially longer (66 days) compared to mice subjected to scrambled control cells (54 days), p=0.052. Conclusion- In summary we show that PHF19 is upregulated in malignant plasma cells of MM patients and that PHF19 expression levels increase with advanced MM stages. High PHF19 expression was a marker of adverse prognosis in our total therapy (TT 3-5) cohort. Most importantly, in-vitro and in-vivo functional studies showed that PHF19 has important biological functions in MM. These results suggest that epigenetic regulation through histone methylation, in particular, H3K27 trimethylation, plays a crucial role in MM and the affected downstream pathways should be further elucidated. Disclosures Boyle: Janssen: Honoraria, Other: Travel; Abbvie: Honoraria; Amgen: Honoraria, Other: travel; Takeda: Honoraria, Other: travel; Celgene Corporation: Honoraria, Other: Travel. van Rhee:Kite Pharma: Consultancy; Adicet Bio: Consultancy; Karyopharm Therapeutics: Consultancy; Takeda: Consultancy; Sanofi Genzyme: Consultancy; Castleman Disease Collaborative Network: Consultancy; EUSA: Consultancy. Walker:Celgene: Research Funding.

scholarly journals Nucleotide Excision Repair (NER) Is Frequently Impaired and Affects Outcome in Multiple Myeloma (MM)

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2055-2055
Author(s):  
Raphael Szalat ◽  
Matija Dreze ◽  
Mehmet Kemal Samur ◽  
Anne S. Calkins ◽  
Giovanni Parmigiani ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Dna Repair ◽  
Genomic Instability ◽  
Nucleotide Excision Repair ◽  
Cell Lines ◽  
Excision Repair ◽  
Sequencing Data ◽  
Nucleotide Excision

Abstract Introduction Multiple Myeloma (MM) is a heterogeneous disease characterized by genomic instability and eventual poor outcome. Aberrations in DNA repair-related pathways have been considered to explain the instability. Nucleotide excision repair (NER) is an important pathway involved in the removal of bulky adducts and DNA crosslinks induced by various genotoxins. Little is known about the relationship between NER in MM biology and patient outcomes. Here we assess the role of NER in MM. Methods We evaluated NER efficiency in a panel of MM cell lines (n=18), with a functional assay based on the purified DNA-Damage Binding protein 2 (DDB2) complex (DDB2 proteo-probe, Dreze et al. 2014). NER proficiency was correlated with cytogenetic characteristics, p53 status, sequencing data, gene expression profile, and with melphalan (MLP) sensitivity evaluated by CellTiterGlo (CTG). We then evaluated NER efficiency in patient samples and interrogated the role of NER in MM patients by correlating expression of NER genes with survival (OS) in a cohort of 170 patients (IFM 2005-01) homogeneously treated with alkylating agents. Results NER, measured as the amount of (6-4) photoproducts remaining 2 hours after UV irradiation, showed variability between MM cell lines. Out of 18 cell lines, 7 exhibited various levels of NER deficiencies, defined as less than 90% repair at 2 hours (4 cell lines 90-70% and 3 cell lines <60%). The other 11 cell lines presented more than 90% of repair. P53 loss of function did not associate with NER deficiency. Notably, all t(4;14) cell lines tested (n=5) showed a NER repair rate > 90%. NER deficient cell lines (NER <90%) were sensitive to melphalan. However all melphalan sensitive cells did not exhibit NER deficiency, This suggests that other DNA repair pathways are involved in the repair of melphalan-induced lesions. Furthermore, we performed the assay in patient samples showing variable levels of NER, which may reflect different disease status and prognosis. Whole genome sequencing data from 6 NER deficient cell lines revealed missense mutations in critical NER genes in 2 of these cell lines. MM1S and MM1R cells showed mutations in the Xeroderma Pigmentosum Complementation Group A (XPA) gene (mutation D70H), and MM1R was also mutated in the Cockayne syndrome, ERCC6 gene (mutation L682I). Gene expression profile comparison in 12 of these showed a positive correlation between expression of NER genes and NER efficiency. We next studied expression of 20 NER genes in 170 patients treated with high dose melphalan (IFM 2005-01). The analysis revealed a significant negative correlation between 5 overexpressed NER genes (ERCC3, ERCC4, ERCC6, MMS19 and NTHL1) and overall survival (OS). Conclusion NER efficiency is heterogeneous in MM, in part due to acquired mutations. Impairment of NER is associated with outcome as well as may contribute to genomic instability. Ability to proficiently measure NER in patient samples provides us an opportunity to now evaluate NER as a prognostic marker in myeloma. Disclosures No relevant conflicts of interest to declare.

scholarly journals HDAC8 Maintain Cytoskeleton Integrity Via Homologous Recombination and Represent a Novel Therapeutic Target in Multiple Myeloma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4385-4385
Author(s):  
Zuzana Chyra ◽  
Maria Gkotzamanidou ◽  
Masood A. Shammas ◽  
Vassilis L. Souliotis ◽  
Yan Xu ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Dna Repair ◽  
Homologous Recombination ◽  
Cell Lines ◽  
Hdac Inhibitors ◽  
Mass Spectrometry Analysis ◽  
Dependent Manner ◽  
Large Panel ◽  
Dose Dependent

Multiple Myeloma (MM) is a plasma cell malignancy vulnerable to epigenetic intervention, with histone deacetylases (HDACs) emerging as the most promising epigenetic targets in combination with current anti-myeloma agents. Pan-HDAC inhibitors are effective as therapeutic agents both in preclinical and clinical setting; however, there is an increasing emphasis on understanding the biological and molecular roles of individual HDACs to limit toxicities observed with pan-HDAC inhibitors. Based on correlation with patient outcome in three independent myeloma datasets, we have evaluated the functional role of HDAC8, a member of Class I HDAC isoenzymes, in MM. Unlike other isoforms, there is limited information about molecular and epigenomic functions of HDAC8. We have previously confirmed expression of HDAC8 in a large panel of MM cell lines, where it is localized predominantly to cytoplasm. Moreover, genetic and pharmacological modulation of HDAC8 with RNAi and specific inhibitor PCI-34051 resulted in a significant inhibition of myeloma cell proliferation and decrease in colony formation (p<.001). HDAC8 inhibition led to an increase in the ongoing spontaneous and radiation-induced DNA damage in MM cells by affecting DNA repair via the homologous recombination (HR) pathway, suggesting a novel function of HDAC8 in promoting HR and DNA repair in MM cells. Using laser micro-irradiation in MM1S and U2OS cells, we observed HDAC8 recruitment to DSBs sites and its co-localization with Rad51 and Scm3, a member of cohesin complex. A transcriptomic analysis of HDAC8 knock-down cells also shows perturbation of number of cytoskeleton-related genes confirming significant role of HAD8 in cytoskeleton rearrangement in MM. Mass-spectrometry analysis to identify the HDAC8 substrates in MM cells is currently ongoing. Classical pan-HDACi, such as SAHA (vorinostat), bind to HDAC8 with substantially diminished activity (IC50 = 2 μM), reflecting a unique binding site of this isoform. To discover and validate new small molecules with HDAC8 subtype selectivity, we have explored the efficacy of OJI-1, a novel selective and potent HDAC8 inhibitor (IC50 = 0.8 nM) with modest inhibition of HDAC6 (1200 nM). Treatment with OJI-1 selectively impact cell viability of a large panel of MM cell lines (n=20) in a time and dose dependent manner, while sparing healthy donors PBMC both in resting and activated state (n=3). The significantly higher IC50 observed in PBMCs suggests a favorable therapeutic index. Western blotting analysis confirmed target selectivity with significant time and dose dependent decrease in H3 and H4 acetylation in MM cells treated with OJI-1. Moreover, pharmacological inhibition of HDAC8 specifically inhibited HR but not non-homologous end joining. These data suggest that targeting of HDAC8 using OJI-1 could be effective treatment approach in MM. Based on molecular data combination studies and in vivo evaluation are ongoing. In conclusion, our results provide insight into the role of HDAC8 in DNA stability and cell growth and viability which can be exploited in future for therapeutic application alone and in combination in MM. Disclosures Munshi: Takeda: Consultancy; Janssen: Consultancy; Amgen: Consultancy; Abbvie: Consultancy; Janssen: Consultancy; Celgene: Consultancy; Takeda: Consultancy; Adaptive: Consultancy; Amgen: Consultancy; Adaptive: Consultancy; Abbvie: Consultancy; Oncopep: Consultancy; Oncopep: Consultancy; Celgene: Consultancy.

scholarly journals Differential epigenetic regulation between the alternative promoters, PRDM1α and PRDM1β, of the tumour suppressor gene PRDM1 in human multiple myeloma cells

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Raquel Romero-García ◽  
Laura Gómez-Jaramillo ◽  
Rosa María Mateos ◽  
Gema Jiménez-Gómez ◽  
Nuria Pedreño-Horrillo ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Plasma Cells ◽  
Regulatory Elements ◽  
Suppressor Gene ◽  
Tumour Suppressor Gene ◽  
Alternative Promoters ◽  
Therapeutic Approaches ◽  
Human Multiple Myeloma

Abstract Multiple myeloma (MM) is a B-cell neoplasm that is characterized by the accumulation of malignant plasma cells in the bone marrow. The transcription factor PRDM1 is a master regulator of plasma cell development and is considered to be an oncosuppressor in several lymphoid neoplasms. The PRDM1β isoform is an alternative promoter of the PRDM1 gene that may interfere with the normal role of the PRDM1α isoform. To explain the induction of the PRDM1β isoform in MM and to offer potential therapeutic strategies to modulate its expression, we characterized the cis regulatory elements and epigenetic status of its promoter. We observed unexpected patterns of hypermethylation and hypomethylation at the PRDM1α and PRDM1β promoters, respectively, and prominent H3K4me1 and H3K9me2 enrichment at the PRDM1β promoter in non-expressing cell lines compared to PRDM1β-expressing cell lines. After treatment with drugs that inhibit DNA methylation, we were able to modify the activity of the PRDM1β promoter but not that of the PRDM1α promoter. Epigenetic drugs may offer the ability to control the expression of the PRDM1α/PRDM1β promoters as components of novel therapeutic approaches.

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